Methods for diagnosing, prognosing and treating muscular dystrophy

ABSTRACT

Disclosed herein are methods for diagnosing, prognosing and treating muscular dystrophy. Also disclosed are methods of determining the effectiveness of an agent for the treatment of muscular dystrophy. Provided are methods of enhancing muscle regeneration, repair, or maintenance in a subject by administering galectin, such as Galectin-1 and/or Galectin-3 to a subject in need thereof. Also disclosed are methods of increasing or maintaining muscle strength and/or bone density in a subject by administering an effective amount of a Galectin-1 composition, Galectin-3 composition or a combination thereof to the subject in need thereof. Methods of preventing, inhibiting and/or reducing muscle loss and/or bone loss in a subject by administering an effective amount of a Galectin-1 composition, Galectin-3 composition or a combination thereof to the subject in need thereof are disclosed.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. application Ser. No.13/572,508, filed Aug. 10, 2012, which claims benefit to U.S.Provisional Application No. 61/522,507, filed Aug. 11, 2011, whichapplications are incorporated herein in their entirety.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made with government support under R01 AR053697 andR21 NS58429 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

FIELD

This disclosure relates to the field of muscular dystrophy and inparticular, to methods for diagnosing, prognosing and treating patientswith muscular dystrophy, such as merosin deficient congenital musculardystrophy Type 1A, limb-girdle muscular dystrophy, facioscapulohumeralmuscular dystrophy, Beckers muscular dystrophy and Duchenne musculardystrophy.

BACKGROUND

The muscular dystrophies are a group of diverse, heritable neuromusculardisorders which represent a group of devastating neuromuscular diseasescharacterized by primary or secondary skeletal muscle involvement.Duchenne muscular dystrophy (DMD) is an X-chromosome-linked disease andthe most common form of muscular dystrophy. DMD affects 1 in 3500 livemale births with patients suffering from chronic muscle degeneration andweakness. Clinical symptoms are first detected between the ages of 2 and5 years and, by the time the patient is in their teens, the ability forindependent ambulation is lost. Death typically occurs in the patientbefore they are 30 years old due to cardiopulmonary failure.

Congenital muscular dystrophy (CMD) refers to a group of heritableneuromuscular disorders characterized by muscle weakness at birth or ininfancy. Affected infants will present with poor muscle tone and fewmovements. The quality of life and life span of the child is affectedthrough progressive muscle wasting, respiratory compromise, and spinalrigidity. Merosin deficient congenital muscular dystrophy (MDC1A) is themost common and severe form of congenital muscular dystrophy, accountingfor 30-40% of all CMD diagnosed cases. MDC1A is characterized bycongenital hypotonia, distinct joint contractures, and a lack ofindependent ambulation. Feeding tube placement and positive pressureventilation is often required for the respiratory problems that occur.MDC1A has no cure and patients often die before they reach the age often years. Currently there is no cure for either DMD or MDC1A.

SUMMARY

Muscular dystrophies including MDC1A, DMD, Limb-Girdle musculardystrophy (LGMD), facioscapulohumeral muscular dystrophy (FHMD), Beckersmuscular dystrophy are devastating neuromuscular diseases. In additionto there being no cure for such diseases, there are no non-invasivemethods of diagnosing, prognosing or evaluating the efficacy oftreatments for such conditions. Currently, serum creatine kinase levelsand fine needle biopsies are used as tests for DMD, LGMD, FMD, Beckersmuscular dystrophy and MDC1A. However, muscle biopsies are painful,invasive and impractical to perform consistently, and serum creatinekinase levels can vary from day to day in the same patient making, themunreliable indicators of change. A biomarker which can be monitoredeasily, such as in serum or urine, and that can reliably indicatedisease progression is needed.

Disclosed herein are muscular dystrophy-associated molecules that can beused as biomarkers to diagnose and/or prognose muscular dystrophy,including DMD, LGMD, FHMD, Beckers muscular dystrophy and/or MDC1A. Insome embodiments, the muscular dystrophy-associated molecules caninclude, consist essentially of, or consist of disintegrin andmetalloproteinase with thrombospondin motifs 5 (Adamts5), agrin (Agrn),collagen 6A1 (Col6a1), Galectin-1, Galectin-3, matrix metalloproteinase2 (Mmp2), integrin α3 (Iga3), integrin α6 (Iga6), integrin α7 (Iga7),laminin-α4 (Lama4), laminin-α5 (Lama5), nidogen 1 (Nid1), tenascin C(Tnc), tissue inhibitor of metalloproteinase 1 (Timp1), tissue inhibitorof metalloproteinase 2 (Timp2) or any combination thereof. In someexamples, muscular dystrophy-associated molecules include Galectin-1,Galectin-3, Col6A1, Itga3, Iga6, Itga7, Tnc and Timp 1. In someexamples, muscular dystrophy-associated molecules include Galectin-1 andGalectin-3. In some examples, muscular dystrophy-associated moleculesinclude Galectin-3 and Tnc. In some examples, the musculardystrophy-associated molecules include at least Galectin-3 for detectingDMD, LGMD, FHMD, Beckers muscular dystrophy or MDC1A. In some examples,the muscular dystrophy-associated molecules include at least Galectin-3for detecting DMD.

Also disclosed herein are methods of diagnosing or prognosing a subjectwith muscular dystrophy. In some examples, the method includes detectingat least one of the disclosed muscular dystrophy-associated molecules ina sample obtained from the subject at risk of having or having one ormore signs or symptoms associated with muscular dystrophy, therebydiagnosing or prognosing the subject with muscular dystrophy. In someexamples, the method further includes comparing expression of Galectin-1or Galectin-3 in the sample obtained from the subject at risk of havingor having one or more signs or symptoms associated with musculardystrophy to a control, wherein increased expression of Galectin-1 orGalectin-3 molecules relative to a control indicates that the subjecthas muscular dystrophy.

Further, methods of determining the effectiveness of an agent for thetreatment of muscular dystrophy in a subject with muscular dystrophy aredisclosed. In some examples, the methods include detecting expression ofa muscular dystrophy-associated molecule, such as Galectin-3, in asample from the subject following treatment with the agent; andcomparing expression of the muscular dystrophy-associated molecule, suchas Galectin-3, following treatment to a reference value, wherein analteration in the expression of the muscular dystrophy-associatedmolecule, such as Galectin-3, following treatment indicates that theagent may be effective for the treatment of muscular dystrophy in thesubject.

Also disclosed are methods of treating muscular dystrophy. In someexamples, the method includes administering to the subject with musculardystrophy an effective amount of an agent that alters the expression orbiological activity of a muscular dystrophy-associated molecule therebytreating the muscular dystrophy and increasing the subject's chance ofsurvival or delaying the onset of one or more signs or symptomsassociated with the muscular dystrophy. Depending upon the agent, thealteration may be a decrease or an increase.

Methods of treating a subject with galectin or a composition thatincludes galectin are also disclosed. For example, some embodimentsprovide methods of improving muscular health, such as enhancing muscleregeneration, maintenance, or repair in a subject by administering tothe subject an effective amount of galectin or a composition comprisinggalectin, including fragments, derivatives, or analogs thereof.

In some embodiments, methods of increasing/maintaining muscle strengthand/or bone density are provided. In some embodiments, methods ofpreventing, inhibiting and/or reducing muscle and/or bone loss areprovided. In some examples, a galectin composition, such as galectin-1composition, is administered to increase muscle strength and/or bonedensity and/or prevent, inhibit or slow muscle and bone loss. In someexamples, a galectin composition, such as galectin-1 composition, isadministered to a subject at risk of muscle/bone injury or muscle/boneloss, such as to an athlete, an astronaut, or any other individual thatpartakes in activities that may cause muscle/bone injury and/or loss. Insome examples, a disclosed regimen, such as a disclosed Galectin-1regimen, is provided to prevent muscle/bone injury and/or loss. In someexamples, a disclosed regimen, such as a disclosed Galectin-1 regimen,is provided to maintain bone density and/or muscle strength. In someexamples, a disclosed regimen, such as a disclosed Galectin-1 regimen,is provided to treat a subject experiencing a loss in bone densityand/or muscle strength whereby the regimen is administered in a mannerto lead to an increase in bone density and/or muscle strength or tomaintain the existing muscle strength and bone density (e.g., preventfurther loss of muscle strength and/or bone density).

In some examples, a galectin composition, such as a Galectin-1composition, is administered to a subject at risk of acquiring orsuffering from a condition or disease associated with muscle loss, boneloss, muscle density loss, and/or muscle strength loss, such as, but notlimited to a subject at risk of acquiring or suffering from kyphosis,muscular dystrophies, broken bones, muscle strains, muscle tears, tendoninjury, osteoporosis, rheumatoid arthritis, lupus, scoliosis and/ormultiple sclerosis. In some examples, a galectin composition, such as aGalectin-1 composition, is administered for preventing, treating orslowing the progression of a sign or symptom associated with aging. Itis contemplated that a galectin composition can be administered forshort or prolong periods of time, ranging from days to years. It iscontemplated that a galectin composition can be administered as anutritional supplemental.

In some examples, a galectin composition, such as Galectin-1, isadministered post-surgery, such as to a subject that has undergonesurgery and may be at risk of experiencing or has muscle loss, boneloss, muscle strength loss or muscle density loss. In some examples, agalectin composition, such as Galectin-1, is administered to a femalesubject post-pregnancy. In some examples, a galectin composition, suchas Galectin-1, is administered to a short or long-term coma subject.

In a specific example, the galectin is a complete galectin protein, suchas Galectin-1 or Galectin-3. In further examples, the galectin isselected from Galectin-1, Galectin-3, and combinations thereof. Infurther examples, the galectin or galectin composition includes asubstance at least substantially homologous to Galectin-1 or Galectin-3.In yet further implementations, the galectin or galectin compositioncomprises a polypeptide at least substantially homologous to theGalectin-1 or Galectin-3.

In additional examples, the galectin or galectin composition consists ofGalectin-1, Galectin-3, and combinations thereof. In further examples,the galectin or galectin composition consists of a substance at leastsubstantially homologous to Galectin-1 or Galectin-3. In a specificexample, the galectin or galectin composition does not include agalectin fragment, such as including only a complete galectin protein.

In yet another example, the galectin or galectin composition consistsessentially of Galectin-1, Galectin-3, and combinations thereof. Infurther examples, the galectin or galectin composition consistsessentially of a substance at least substantially homologous toGalectin-1 or Galectin-3. In yet further implementations, the galectinor galectin composition consists essentially of a polypeptide at leastsubstantially homologous to the galectin α1 chain. In a specificexample, the galectin or galectin composition does not include agalectin fragment, such as including essentially only a completegalectin protein.

Further implementations of the disclosed method include diagnosing thesubject as having a condition treatable by administering galectin or acomposition comprising galectin, such as by administering Galectin-1,Galectin-3 or a combination thereof or a composition containing

Galectin-1, Galectin-3 or a combination. In one example, the subject isdiagnosed as suffering from muscular dystrophy, such as LGMD, FHMD,Beckers muscular dystrophy and/or MDC1A. In further instances thecondition is characterized by the failure of a subject, or the reducedability of the subject, to express one or more proteins associated withthe formation or maintenance of the extracellular matrix, such asimpaired or non-production of a galectin, an integrin, dystrophin,utrophin, or dystroglycan.

In a specific embodiment, the present disclosure also provides a methodfor increasing muscle regeneration in a subject by administering aneffective amount of a galectin composition, such as an effect amount ofa Galectin-1 and/or Galectin-3 composition. For example, geriatricsubjects, subjects suffering from muscle disorders, and subjectssuffering from muscle injury, including activity induced muscle injury,such as injury caused by exercise, may benefit from this embodiment.

In yet further embodiments of the disclosed method, the galectin orgalectin composition, such as Galectin-1, Galectin-3 or a combinationthereof containing composition, is administered in a preventativemanner, such as to prevent or reduce muscular or bone damage or injury(such as activity or exercise induced injury). For example, geriatricsubjects, post-surgery subjects, post-pregnancy, subjects prone tomuscle damage, or subjects at risk for muscular injury, such asathletes, may be treated in order to prevent, eliminate or amelioratebone or muscular damage, injury, or disease.

Implementations of the present disclosure may also be used to promotewound healing. In some examples, a galectin or a composition comprisinggalectin is administered into or proximate to a wound. In furtherexamples, the substance is administered systemically. Although thesubstance is typically applied after the wound occurs, the substance isapplied prospectively in some examples.

In further embodiments, the method of the present disclosure includesadministering the galectin or galectin composition, such as Galectin-1,Galectin-3 or a combination thereof containing composition, with one ormore additional pharmacological substances, such as a therapeutic agent.In some aspects, the additional therapeutic agent enhances thetherapeutic effect of the galectin or galectin composition. In furtheraspects, the therapeutic agent provides independent therapeutic benefitfor the condition being treated. In various examples, the additionaltherapeutic agent is a component of the extracellular matrix, such as anintegrin, dystrophin, dystroglycan, utrophin, or a growth factor. Infurther examples, the therapeutic agent reduces or enhances expressionof a substance that enhances the formation or maintenance of theextracellular matrix.

In some examples, the galectin or galectin composition is applied to aparticular area of the subject to be treated. For example, the galectinor galectin composition may be injected into a particular area to betreated, such as a muscle. In further examples, the galectin or galectincomposition is administered such that it is distributed to multipleareas of the subject, such as systemic administration or regionaladministration.

Galectin, or a composition comprising galectin, such as Galectin-1,Galectin-3, or a combination thereof, can be administered by anysuitable method, such as topically, parenterally (such as intravenouslyor intraperitoneally), or orally. In a specific example, the galectin orgalectin composition is administered systemically, such as throughparenteral administration, such as stomach injection or peritonealinjection.

Although the disclosed methods generally have been described withrespect to muscle regeneration, the disclosed methods also may be usedto enhance repair or maintenance, or prevent damage to, other tissuesand organs. For example, the methods of the present disclosure can beused to treat symptoms of muscular dystrophy stemming from effects tocells or tissue other than skeletal muscle, such as impaired or alteredbrain function, smooth muscles, or cardiac muscles.

Methods of identifying agents for use in treating muscular dystrophy arealso provided. In some examples, the method includes contacting a samplewith one or more test agents under conditions sufficient for the one ormore test agents to alter the activity of a musculardystrophy-associated associated molecule, such as Galectin-1 orGalectin-3; detecting activity of the muscular dystrophy-associatedmolecule, such as Galectin-1 or Galectin-3, in the presence of the oneor more test agents; and comparing activity of musculardystrophy-associated molecule, such as Galectin-1 or Galectin-3, in thepresence of the one or more test agents to a reference value todetermine if there is an alteration in expression of the musculardystrophy-associated molecule, such as Galectin-1 or Galectin-3, whereinaltered expression of the muscular dystrophy-associated molecule, suchas Galectin-1 or Galectin-3, indicates that the one or more test agentsmay be of use to treat the muscular dystrophy. In some examples, anincrease in Galectin-1 indicates that the test agent can be used totreat muscular dystrophy.

The foregoing and other features of the disclosure will become moreapparent from the following detailed description, which proceeds withreference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A and 1B are bar graphs illustrating transcription of Lgals1 andLgals3 are altered in the dy^(W) −/− mouse. FIG. 1A shows the transcriptfor Lgals1 (Galectin-1) was significantly increased over wild-type inboth 4- and 8-week old animals. FIG. 1B illustrated that the transcriptfor Lgals3 (Galectin-3) was also significantly increased over wild-typein both 4- and 8-week old animals. **P<0.01, ****P<0.00001.

FIGS. 2A-2C are digital images and bar graphs illustrating Westernblotting studies for Galectin-1 in the dy^(W) −/− and wild-type mice at4- and 8-weeks of age. FIG. 2A shows the difference in Galectin-1protein in the muscles of 4-week old dy^(W) −/− mice was significantlydifferent from wild-type animals. FIG. 2B indicates that the level ofGalectin-1 protein in the grostocnemius muscle of 8-week old dy^(W) −/−animals was not significantly different from that measured in wild-type.FIG. 2C shows that there was no difference in Galectin-1 protein indy^(W) −/− mice at 4- and 8-weeks of age.

FIG. 3A is a digital image of Western blotting results for Galectin-3 inthe dy^(W) −/− and wild-type mice at 4- and 8-weeks of age.

FIG. 3B is a bar graph quantitating the level of Galectin-3 protein inthe grostocnemius muscle of 4-week old and 8-week old dy^(W) −/− animalsas compared to wild type (control) mice.

FIGS. 4A and 4B are digital images and bar graphs illustrating Westernblotting studies for Galectin-3 in the serum dy^(W) −/− and wild-typemice at 4- and 8-weeks of age. FIG. 4A shows that there was nosignificant difference in Galectin-3 protein in the serum of 4-week olddy^(W) −/− mice when compared to wild-type animals. FIG. 4B shows thatthere was no difference in Galectin-3 protein in dy^(W) −/− mice serumat 4- and 8-weeks of age.

FIG. 5 is a series of digital images of Galectin-3 immunofluorescence on4- and 8-week dy^(W) −/− and wild-type mice. Immunofluorescence was usedto evaluate Galectin-3 levels in the tibialis anterior muscle of mice.Galectin-3 was found to be elevated in 4-week old dy^(W) −/− mice whencompared to that in the wild-type mice. Galectin-3 was found to besimilar in 8-week dy^(W) −/− mice and wild-type mice. Galectin-3 levelswere also found to be similar between 4- and 8-week old dy^(W) −/− mice.Galectin-3 levels appear to increase in the wild-type mice as they age.

FIGS. 6A and 6B are bar graphs illustrating transcription of Lgals1 andLgals3 were altered in the mdx mouse. FIG. 6A shows the transcript forLgals1 (Galectin-1) is significantly increased over wild-type in boththe 5- and 10-week old animals. FIG. 6B illustrates the transcript forLgals3 (Galectin-3) was significantly increased over wild-type in both5- and 10-week old animals.

FIGS. 7A-7C are bar graphs and digital images of Western blottingresults for Galectin-1 in mdx and wild-type mice at 2-, 5- and 10-weeksof age. FIG. 7A indicates no significant difference was observed inGalectin-1 protein in the muscles of 5-week old mdx mice when comparedto wild-type animals. FIG. 7B illustrates that the level of Galectin-1protein in the gastrocnemius muscle of 5-week old mdx animals comparedto wild-type were not significantly different. FIG. 7C. shows there wasno difference between Galectin-1 protein in the mdx mice at 2- and5-weeks of age. There was a significant difference between the 2- and10-week old mice and the 5- and 10-week old mice.

FIGS. 8A and 8B are bar graphs and digital images of Western blottingresults for Galectin-3 in the mdx and wild-type mice at 5- and/or10-weeks of age. FIG. 8A shows a significant difference in Galectin-3protein in the muscles of 5-week old mdx mice when compared to wild-typeanimals. FIG. 8B indicates that the level of Galectin-3 protein in thegastrocnemius muscle of 5- and 10-weeks of age mdx and wild-type miceand demonstrates that Galectin-3 protein in 10-week old mdx animals wassignificantly greater than Galectin-3 levels in 5-week old mdx animals.

FIGS. 9A and 9B are bar graphs and digital images of Western blottingstudies for Galectin-3 in the serum of mdx and wild-type mice at 5- and10-weeks of age. FIG. 9A shows there was no significant difference inGalectin-3 protein in the serum of 5-week old mdx mice when compared towild-type animals. FIG. 9B indicates there was no significant differencein Galectin-3 protein between 10-week old mdx mice serum and age-matchedwild-type serum.

FIG. 10 is a series of digital images of Galectin-3 immunofluorescenceon 5- and 10-week mdx and wild-type mice. Immunofluorescence was used toevaluate Galectin-3 levels in the tibialis anterior muscle. Galectin-3was found to be elevated in 5- and 10-week old mdx mice when compared tothat in the wild-type mice. Galectin-3 was found to be elevated in10-week old mdx mice compared to that in the 5-week old mice whileGalectin-3 levels were similar between 5- and 10-week wild-type mice.

FIG. 11 is a digital image of a Western blot study for Galectin-3 levelsin the muscle of the golden retriever muscular dystrophy (GRMD) dogmodel of DMD. Elevated levels of Galectin-3 protein are detected in themuscle of GRMD dogs, lanes A and E. Little or no Galectin-3 was observedin unaffected control dog samples, lanes B-D.

FIG. 12 is a digital image of Galectin-1 fractions eluted from Talonaffinity columns.

FIG. 13 is a graph and table illustrating Galectin-1 treatment decreasesmuscle damage in mdx mice.

FIG. 14 is a graph, table and digital image illustrating Galectin-1treatment increases α7 integrin.

FIGS. 15A-15D demonstrate Galectin-1 treatment of myoblasts and myotubesleads to elevated levels of a7 and b1 Integrins at both the transcriptand protein levels. FIG. 15A is a bar graph illustrating the effect oftreating a7^(+/bGal) myoblasts with increasing levels of recombinantGalectin-1 and then examining for an increase in β-Galactosidaseactivity (a reporter for ITGA7 expression levels) using an FDG activityassay. FIG. 15B is a bar graph illustrating the effect of treating C2C12myoblasts with Galectin-1 or PBS and then examining for levels of α7Integrin protein relative to GAPDH levels, quantitated and graphed. FIG.15C is a bar graph illustrating the effect of treating C2C12 myoblastswith Galectin-1 or PBS and then examining for levels of β1 Integrinprotein levels relative to Ponceau S stain, quantitated and graphed.FIG. 15D is a bar graph illustrating the quantitative real-time PCRresults for ITGA7, ITGB1, and LGALS1 levels from C2C12 myoblasts andmyotubes treated with PBS or 0.2 mM recombinant Galectin-1.

FIGS. 16A-16D are bar graphs illustrating intermuscular (IM) injectionsof mdx mouse tibialus anterior (TA) muscles with recombinant Galectin-1reduces muscle damage and the need for regeneration as determined by thehistological appearance of centrally located nuclei (CLN). Mdx mouse TAmuscles were IM injected with 20 ng (FIGS. 16A and 16B), 1.5 mg (FIG.16C), or 150 mg (FIG. 16D) of recombinant Galectin-1 protein at 4, 3, 5,and 5 weeks old, respectively. Tissues were dissected 48 hourspost-injection, cryosectioned, stained using standard hemotoxylin andeosin procedures and analyzed for CLN. Significance was calculated bystudent t-test (ns=no significance, *p<0.05).

FIGS. 17A-17J are bar graphs illustrating Galectin-1 treatment of mdxmice increases protein levels of members of the sarcolemmal stabilizingdystroglycan complex (DGC) which are normally lost in the absence ofdytrophin. The TA proteins of mdx mice treated weekly by intraperitonealinjections of PBS or 5 mg/kg recombinant Galectin-1 and were examinedusing standard western blotting procedures. Results for α7A Integrin(FIG. 17A), α7B Integrin (FIG. 17B), β1D Integrin (FIG. 17C),α-dystroglycan (FIG. 17D), β-dystroglycan (FIG. 17E), β-sarcoglycan(FIG. 17F), γ-sarcoglycan (FIG. 17G), δ-sarcoglycan (FIG. 17H),ε-sarcoglycan (FIG. 171), and sarcospan (FIG. 17J) were quantified forthe two mouse groups and graphed relative a-tubulin. Significance wascalculated by student t-test (*p<0.05, **p<0.01, ***p<0.001).

FIGS. 18A-18C are bar graphs illustrating galectin-1 treatment of mdxmice increases transcript levels of members of the α7β1 Integrin complexand LGALS1. The TA transcripts of mdx mice treated weekly byintraperitoneal injections of PBS or 5 mg/kg recombinant Galectin-1 andwere analyzed using quantitative real-time PCR. Results for ITGA7 (FIG.18A), ITGB 1 (FIG. 18B), and LGALS1 (FIG. 18C) are shown aftercalculating relative fold compared to GAPDH.

FIGS. 19A-19D are graphs illustrating galectin-1 treatment of mdx miceincreases relative strength, decreases fatigue, and normalizes musclehistological fiber size. (FIG. 19A) Grip strength studies on mdx micetreated weekly by intraperitoneal injections of PBS or 5 mg/kgrecombinant Galectin-1 or untreated black-10 mice were performed usingstandard procedures. Average strength per gram body weight (FIG. 19B)and percent fatigue (FIG. 19C) were then calculated and graphed. (FIG.19D) The TA was cryosectioned into 10 mM sections and fiber sizes weredetermined using the minimum Feret's diameter measurement on H&E stainedsections, Feret's diameter for at least 1000 fibers per group weremeasured. Significance was calculated by student t-test (*p<0.05,**p<0.01, ***p<0.001).

FIG. 20 is a set of digital images and a bar graph showing galectin-1treatment of mdx mice leads to increased muscle strength which preventskyphosis in 10-week old mice. Sagittal X-ray images were taken of PBS(top, n=2) or 5 mg/kg/week recombinant Galectin-1 (bottom, n=4) treatedmdx mice at 10-weeks of age. Spinal curvature (kyphosis) was analyzed bydrawing a line from the base of the spine at the neck to the base of thespine at the beginning of the hip bone. A perpendicular line was thendrawn from the apex of the spinal curve and the length of this line wasused to measure kyphosis. Significance was calculated by student t-test(*p<0.05).

FIGS. 21A-21F illustrate galectin-1 treatment of mdx mice leads toincreased bone growth during development. Sagittal cranium (FIG. 21A,example) and coronal body (FIG. 21B, example) X-ray images were taken ofPBS or 5 mg/kg/week recombinant Galectin-1 treated mdx mice at 10-weeksof age. From these images the lower jaw lengths (FIG. 21C), femurlengths (FIG. 21D), femur area (FIG. 21E), and tibia lengths (FIG. 21F)were measured and graphed. Significance was calculated by student t-test(*p<0.05, **p<0.01, ***p<0.001).

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS I. Overview of SeveralEmbodiments

Disclosed herein are muscular dystrophy-associated molecules that can beused as biomarkers to diagnose and/or prognose muscular dystrophy,including DMD, LGMD, FHMD, Beckers muscular dystrophy and/or MDC1A. Insome embodiments, the muscular dystrophy-associated molecules caninclude, consist essentially of, or consist of disintegrin andmetalloproteinase with thrombospondin motifs 5 (Adamts5), agrin (Agrn),collagen 6A1 (Col6a1), Galectin-1, Galectin-3, matrix metalloproteinase2 (Mmp2), integrin α3 (Iga3), integrin α6 (Iga6), integrin α7 (Iga7),laminin-α4 (Lama4), laminin-α5 (Lama5), nidogen 1 (Nid1), tenascin C(Tnc), tissue inhibitor of metalloproteinase 1 (Timp1), tissue inhibitorof metalloproteinase 2 (Timp2) or any combination thereof. In someexamples, muscular dystrophy-associated molecules include Galectin-1,Galectin-3, Col6A1, Itga3, Iga6, Itga7, Tnc and Timp 1. In someexamples, muscular dystrophy-associated molecules include Galectin-1 andGalectin-3. In some examples, muscular dystrophy-associated moleculesinclude Galectin-3 and Tnc. In some examples, the musculardystrophy-associated molecules include at least Galectin-3 for detectingDMD, LGMD, FHMD, Beckers muscular dystrophy or MDC1A. In some examples,the muscular dystrophy-associated molecules include at least Galectin-3for detecting DMD.

Disclosed herein are methods of diagnosing or prognosing a subject withmuscular dystrophy. In some embodiments, a method of diagnosing orprognosing a subject with muscular dystrophy, comprises detectingexpression of one or more of the disclosed muscular dystrophy-associatedmolecules, such as Galectin-1 or Galectin-3, in a sample obtained fromthe subject at risk of having or having one or more signs or symptomsassociated with muscular dystrophy, thereby diagnosing or prognosing thesubject with muscular dystrophy.

In some embodiments, a method of diagnosing or prognosing a subject withmuscular dystrophy, comprises detecting expression of Galectin-1 orGalectin-3 in a sample obtained from the subject at risk of having orhaving one or more signs or symptoms associated with muscular dystrophy,thereby diagnosing or prognosing the subject with muscular dystrophy. Insome embodiments, the method further comprises comparing expression ofGalectin-1 or Galectin-3 in the sample obtained from the subject at riskof having or having one or more signs or symptoms associated withmuscular dystrophy to a control, wherein increased expression ofGalectin-1 or Galectin-3 molecules relative to a control indicates thatthe subject has muscular dystrophy.

In some embodiments, the muscular dystrophy is MDC1A, LGMD, FHMD,Beckers muscular dystrophy or DMD.

In some embodiments, detecting expression comprises detecting Galectin-3and/or Galectin-1.

In some embodiments, increased expression of Galectin-3 moleculesrelative to a control indicates that the subject has a poor prognosisand a decreased chance of survival.

In some embodiments, the muscular dystrophy is DMD.

In some embodiments, the sample is a blood or urine sample.

In some embodiments, expression is measured by real time quantitativepolymerase chain reaction, microarray analysis or Western blot analysis.

In some embodiments, methods of treating muscular dystrophy in a subjectare disclosed. In some embodiments, the method comprises administeringto the subject with muscular dystrophy an effective amount of an agentthat alters expression or biological activity of Galectin-3, therebytreating one or more signs or symptoms associated with musculardystrophy increasing the subject's chance of survival. In someembodiments, the agent reduces the biological activity of Galectin-3. Insome embodiments, the agent increases the biological activity ofGalectin-3.

In some embodiments, methods of treating muscular dystrophy in a subjectare disclosed. In some embodiments, the method comprises administeringto the subject with muscular dystrophy an effective amount of an agentthat increases expression or biological activity of Galectin-1, therebytreating one or more signs or symptoms associated with musculardystrophy increasing the subject's chance of survival. In someembodiments, the agent increases the biological activity of Galectin-1.

In some embodiments, methods of treating a subject with galectin or acomposition that includes galectin are also disclosed. For example, someembodiments provide methods of improving muscular health, such asenhancing muscle regeneration, maintenance, or repair in a subject byadministering to the subject an effective amount of galectin or acomposition comprising galectin, including fragments, derivatives, oranalogs thereof. In a specific example, the galectin is a completegalectin protein. In further examples, the galectin is selected fromGalectin-1, Galectin-3, and combinations thereof. In further examples,the galectin or galectin composition includes a substance at leastsubstantially homologous to Galectin-1 or Galectin-3. In yet furtherimplementations, the galectin or galectin composition comprises apolypeptide at least substantially homologous to the Galectin-1 orGalectin-3.

In some embodiments, methods of increasing/maintaining muscle strengthand/or bone density are provided. Also disclosed herein are methods ofpreventing, inhibiting and/or reducing muscle and/or bone loss. In someexamples, a galectin composition, such as galectin-1 composition, isadministered to increase muscle strength and/or bone density and/orprevent, inhibit or slow muscle and bone loss. In some examples, agalectin composition, such as galectin-1 composition, is administered toa subject at risk of muscle/bone injury or muscle/bone loss, such as toan athlete, an astronaut, or any other individual that partakes inactivities that may cause muscle/bone injury and/or loss. In someexamples, a disclosed regimen, such as a disclosed Galectin-1 regimen,is provided to prevent muscle/bone injury and/or loss. In some examples,a disclosed regimen, such as a disclosed Galectin-1 regimen, is providedto maintain bone density and/or muscle strength. In some examples, adisclosed regimen, such as a disclosed Galectin-1 regimen, is providedto treat a subject experiencing a loss in bone density and/or musclestrength whereby the regimen is administered in a manner to lead to anincrease in bone density and/or muscle strength or to maintain theexisting muscle strength and bone density (e.g., prevent further loss ofmuscle strength and/or bone density).

In some examples, a galectin composition, such as a Galectin-1composition, is administered to a subject at risk of acquiring orsuffering from a condition or disease associated with muscle loss, boneloss, muscle density loss, and/or muscle strength loss, such as, but notlimited to a subject at risk of acquiring or suffering from kyphosis,muscular dystrophies, broken bones, muscle strains, muscle tears, tendoninjury, osteoporosis, rheumatoid arthritis, lupus, scoliosis, and/ormultiple sclerosis. In some examples, a galectin composition, such as aGalectin-1 composition, is administered for preventing, treating orslowing the progression of a sign or symptom associated with aging. Itis contemplated that a galectin composition can be administered forshort or prolong periods of time, ranging from days to years. In someexamples, a galectin composition, such as Galectin-1, is administeredpost-surgery, such as to a subject that has undergone surgery and may beat risk of experiencing or has muscle loss, bone loss, muscle strengthloss or muscle density loss. In some examples, a galectin composition,such as Galectin-1, is administered to a female subject post-pregnancy.In some examples, a galectin composition, such as Galectin-1, isadministered to a short or long-term coma subject.

II. Terms

The following explanations of terms and methods are provided to betterdescribe the present disclosure and to guide those of ordinary skill inthe art in the practice of the present disclosure. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. The term“comprises” means “includes.” Thus, “comprising A or B,” means“including A, B, or A and B,” without excluding additional elements.

It is further to be understood that all base sizes or amino acid sizes,and all molecular weight or molecular mass values, given for nucleicacids or polypeptides are approximate, and are provided for description.Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of this disclosure,suitable methods and materials are described below.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. Definitions of commonterms in molecular biology may be found in Benjamin Lewin, Genes V,published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrewet al. (eds.), The Encyclopedia of Molecular Biology, published byBlackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers(ed.), Molecular Biology and Biotechnology: a Comprehensive DeskReference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Allsequences provided in the disclosed Genbank Accession numbers areincorporated herein by reference as available on Aug. 11, 2011. In caseof conflict, the present specification, including explanations of terms,will control. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

In order to facilitate review of the various embodiments of thisdisclosure, the following explanations of specific terms are provided:

Administration: To provide or give a subject an agent by any effectiveroute. Exemplary routes of administration include, but are not limitedto, injection (such as subcutaneous, intramuscular, intradermal,intraperitoneal, and intravenous), oral, sublingual, rectal,transdermal, intranasal, vaginal and inhalation routes. For example,Galectin, or compositions thereof, also may be administered to a subjectusing a combination of these techniques.

Suitable solid or liquid pharmaceutical preparation forms are, forexample, aerosols, (micro)capsules, creams, drops, drops or injectablesolution in ampoule form, emulsions, granules, powders, suppositories,suspensions, syrups, tablets, coated tablets, and also preparations withprotracted release of active compounds, in whose preparation excipientsand additives and/or auxiliaries such as binders, coating agents,disintegrants, flavorings, lubricants, solubilizers, sweeteners, orswelling agents are customarily used as described above. Thepharmaceutical compositions are suitable for use in a variety of drugdelivery systems. For a brief review of various methods for drugdelivery, see Langer, “New Methods of Drug Delivery,” Science249:1527-1533 (1990), incorporated by reference herein to the extent notinconsistent with the present disclosure.

Galectin, such as Galectin-1, Galectin-3 or the disclosed compositionsor other therapeutic or nutraceutical agents of the present disclosurecan be formulated into active pharmaceutical or nutraceuticalcompositions that can be administered to a subject parenterally ororally. Parenteral administration routes include, but are not limited toepidermal, intraarterial, intramuscular (IM, and depot IM),intraperitoneal (IP), intravenous (IV), intrasternal injection orinfusion techniques, intranasal (inhalation), intrathecal, injectioninto the stomach, subcutaneous injections (subcutaneous (SQ and depotSQ), transdermal, topical, and ophthalmic.

Galectin, such as Galectin-1, Galectin-3 or the disclosed compositionsor other therapeutic or nutraceutical agent can be mixed or combinedwith suitable pharmaceutically acceptable excipients to preparepharmaceutical compositions. Pharmaceutically acceptableexcipients/carriers include, but are not limited to, alumina, aluminumstearate, buffers (such as phosphates), glycine, ion exchangers (such asto help control release of charged substances), lecithin, partialglyceride mixtures of saturated vegetable fatty acids, potassiumsorbate, serum proteins (such as human serum albumin), sorbic acid,water, salts or electrolytes such as cellulose-based substances,colloidal silica, disodium hydrogen phosphate, magnesium trisilicate,polyacrylates, polyalkylene glycols, such as polyethylene glycol,polyethylene-polyoxypropylene-block polymers, polyvinyl pyrrolidone,potassium hydrogen phosphate, protamine sulfate, group 1 halide saltssuch as sodium chloride, sodium carboxymethylcellulose, waxes, wool fat,and zinc salts, for example. Liposomal suspensions may also be suitableas pharmaceutically acceptable carriers.

Upon mixing or addition of a disclosed composition, or other therapeuticagent, the resulting mixture may be a solid, solution, suspension,emulsion, or the like. These may be prepared according to methods knownto those of ordinary skill in the art. The form of the resulting mixturedepends upon a number of factors, including the intended mode ofadministration and the solubility of the agent in the selected carrier.

Pharmaceutical carriers suitable for administration of galectin, such asGalectin-1, Galectin-3 or the disclosed compositions or othertherapeutic agent include any such carriers known to be suitable for theparticular mode of administration. In addition, galectin, such asGalectin-1, Galectin-3 or the disclosed composition or other therapeuticsubstance can also be mixed with other inactive or active materials thatdo not impair the desired action, or with materials that supplement thedesired action, or have another action.

Methods for solubilizing may be used where the agents exhibitinsufficient solubility in a carrier. Such methods are known andinclude, but are not limited to, dissolution in aqueous sodiumbicarbonate, using cosolvents such as dimethylsulfoxide (DMSO), andusing surfactants such as TWEEN® (ICI Americas, Inc., Wilmington, Del.).

Galectin, such as Galectin-1, Galectin-3 or the disclosed compositionsor other therapeutic agent can be prepared with carriers that protectthem against rapid elimination from the body, such as coatings ortime-release formulations. Such carriers include controlled releaseformulations, such as, but not limited to, microencapsulated deliverysystems. The galectin, such as Galectin-1, Galectin-3 or othertherapeutic agent is included in the pharmaceutically acceptable carrierin an amount sufficient to exert a therapeutically useful effect,typically in an amount to avoid undesired side effects, on the treatedsubject. The therapeutically effective concentration may be determinedempirically by testing the compounds in known in vitro and in vivo modelsystems for the treated condition. For example, mouse models of musculardystrophy may be used to determine effective amounts or concentrationsthat can then be translated to other subjects, such as humans, as knownin the art.

Injectable solutions or suspensions can be formulated, using suitablenon-toxic, parenterally-acceptable diluents or solvents, such as1,3-butanediol, isotonic sodium chloride solution, mannitol, Ringer'ssolution, saline solution, or water; or suitable dispersing or wettingand suspending agents, such as sterile, bland, fixed oils, includingsynthetic mono- or diglycerides, and fatty acids, including oleic acid;a naturally occurring vegetable oil such as coconut oil, cottonseed oil,peanut oil, sesame oil, and the like; glycerine; polyethylene glycol;propylene glycol; or other synthetic solvent; antimicrobial agents suchas benzyl alcohol and methyl parabens; antioxidants such as ascorbicacid and sodium bisulfite; buffers such as acetates, citrates, andphosphates; chelating agents such as ethylenediaminetetraacetic acid(EDTA); agents for the adjustment of tonicity such as sodium chlorideand dextrose; and combinations thereof. Parenteral preparations can beenclosed in ampoules, disposable syringes, or multiple dose vials madeof glass, plastic, or other suitable material. Buffers, preservatives,antioxidants, and the like can be incorporated as required. Whereadministered intravenously, suitable carriers include physiologicalsaline, phosphate-buffered saline (PBS), and solutions containingthickening and solubilizing agents such as glucose, polyethylene glycol,polypropyleneglycol, and mixtures thereof. Liposomal suspensions,including tissue-targeted liposomes, may also be suitable aspharmaceutically acceptable carriers.

For topical application, galectin, such as Galectin-1, Galectin-3 or thedisclosed compositions or other therapeutic agent may be made up into acream, lotion, ointment, solution, or suspension in a suitable aqueousor non-aqueous carrier. Topical application can also be accomplished bytransdermal patches or bandages which include the therapeutic substance.Additives can also be included, e.g., buffers such as sodiummetabisulphite or disodium edetate; preservatives such as bactericidaland fungicidal agents, including phenyl mercuric acetate or nitrate,benzalkonium chloride, or chlorhexidine; and thickening agents, such ashypromellose.

If galectin, such as Galectin-1, Galectin-3 or a disclosed compositionor other therapeutic agent is administered orally as a suspension, thepharmaceutical compositions can be prepared according to techniques wellknown in the art of pharmaceutical formulation and may contain asuspending agent, such as alginic acid or sodium alginate, bulkingagent, such as microcrystalline cellulose, a viscosity enhancer, such asmethylcellulose, and sweeteners/flavoring agents. Oral liquidpreparations can contain conventional additives such as suspendingagents, e.g., gelatin, glucose syrup, hydrogenated edible fats, methylcellulose, sorbitol, and syrup; emulsifying agents, e.g., acacia,lecithin, or sorbitan monooleate; non-aqueous carriers (including edibleoils), e.g., almond oil, fractionated coconut oil, oily esters such asglycerine, propylene glycol, or ethyl alcohol; preservatives such asmethyl or propyl p-hydroxybenzoate or sorbic acid; and, if desired,conventional flavoring or coloring agents. When formulated as immediaterelease tablets, these compositions can contain dicalcium phosphate,lactose, magnesium stearate, microcrystalline cellulose, and starchand/or other binders, diluents, disintegrants, excipients, extenders,and lubricants.

If oral administration is desired, the galectin, such as Galectin-1,Galectin-3 or a disclosed composition, or other therapeutic substancecan be provided in a composition that protects it from the acidicenvironment of the stomach. For example, Galectin-1, Galectin-3 or adisclosed composition, or other therapeutic agent can be formulated withan enteric coating that maintains its integrity in the stomach andreleases the active compound in the intestine. The Galectin-1,Galectin-3 or a disclosed composition, or other therapeutic agent canalso be formulated in combination with an antacid or other suchingredient.

Oral compositions generally include an inert diluent or an ediblecarrier and can be compressed into tablets or enclosed in gelatincapsules. For the purpose of oral therapeutic administration, thegalectin, such as Galectin-1, Galectin-3, or disclosed composition, orother therapeutic substance can be incorporated with excipients and usedin the form of capsules, tablets, or troches. Pharmaceuticallycompatible adjuvant materials or binding agents can be included as partof the composition.

The capsules, pills, tablets, troches, and the like can contain any ofthe following ingredients or compounds of a similar nature: a bindersuch as, but not limited to, acacia, corn starch, gelatin, gumtragacanth, polyvinylpyrrolidone, or sorbitol; a filler such as calciumphosphate, glycine, lactose, microcrystalline cellulose, or starch; adisintegrating agent such as, but not limited to, alginic acid and cornstarch; a lubricant such as, but not limited to, magnesium stearate,polyethylene glycol, silica, or talc; a gildant, such as, but notlimited to, colloidal silicon dioxide; a sweetening agent such assucrose or saccharin; disintegrants such as potato starch; dispersing orwetting agents such as sodium lauryl sulfate; and a flavoring agent suchas peppermint, methyl salicylate, or fruit flavoring.

When the dosage unit form is a capsule, it can contain, in addition tomaterial of the above type, a liquid carrier, such as a fatty oil. Inaddition, dosage unit forms can contain various other materials thatmodify the physical form of the dosage unit, for example, coatings ofsugar and other enteric agents. The galectin, such as Galectin-1,Galectin-3 or disclosed composition, or other therapeutic agent can alsobe administered as a component of an elixir, suspension, syrup, wafer,tea, chewing gum, or the like. A syrup may contain, in addition to theactive compounds, sucrose or glycerin as a sweetening agent and certainpreservatives, dyes and colorings, and flavors.

When administered orally, the compounds can be administered in usualdosage forms for oral administration. These dosage forms include theusual solid unit dosage forms of tablets and capsules as well as liquiddosage forms such as solutions, suspensions, and elixirs. When the soliddosage forms are used, they can be of the sustained release type so thatthe compounds need to be administered less frequently.

Agent: Any protein, nucleic acid molecule (including chemically modifiednucleic acids), compound, antibody, small molecule, organic compound,inorganic compound, or other molecule of interest. Agent can include atherapeutic agent, a diagnostic agent or a pharmaceutical agent. Atherapeutic or pharmaceutical agent is one that alone or together withan additional compound induces the desired response (such as inducing atherapeutic or prophylactic effect when administered to a subject,including treating a subject with a muscular dystrophy).

In some examples, an agent can act directly or indirectly to alter theactivity of Galectin-1 and/or Galectin-3. An example of a therapeuticagent is one that can alter the activity of a gene or gene productassociated with muscular dystrophy, for example as measured by aclinical response (such as an increase survival time or a decrease inone or more signs or symptoms associated with the muscular dystrophy).Therapeutically agents also include organic or other chemical compoundsthat mimic the effects of the therapeutically effective peptide,antibody, or nucleic acid molecule.

A “pharmaceutical agent” is a chemical compound or composition capableof inducing a desired therapeutic or prophylactic effect whenadministered to a subject, alone or in combination with anothertherapeutic agent(s) or pharmaceutically acceptable carriers. In aparticular example, a pharmaceutical agent significantly reduces theexpression and/or activity of a muscular dystrophy associated moleculethereby increasing a subject's survival time.

Antibody: A polypeptide including at least a light chain or heavy chainimmunoglobulin variable region which specifically recognizes and bindsan epitope of an antigen, such as a muscular dystrophy-associatedmolecule or a fragment thereof. Antibodies are composed of a heavy and alight chain, each of which has a variable region, termed the variableheavy (V_(H)) region and the variable light (V_(L)) region. Together,the V_(H) region and the V_(L) region are responsible for binding theantigen recognized by the antibody. Antibodies of the present disclosureinclude those that are specific for a muscular dystrophy-associatedmolecule, such as Galectin-1 or Galectin-3.

The term antibody includes intact immunoglobulins, as well the variantsand portions thereof, such as Fab' fragments, F(ab)'₂ fragments, singlechain Fv proteins (“scFv”), and disulfide stabilized Fv proteins(“dsFv”). A scFv protein is a fusion protein in which a light chainvariable region of an immunoglobulin and a heavy chain variable regionof an immunoglobulin are bound by a linker, while in dsFvs, the chainshave been mutated to introduce a disulfide bond to stabilize theassociation of the chains. The term also includes genetically engineeredforms such as chimeric antibodies (for example, humanized murineantibodies), heteroconjugate antibodies (such as, bispecificantibodies). See also, Pierce Catalog and Handbook, 1994-1995 (PierceChemical Co., Rockford, Ill.); Kuby, J., Immunology, 3rd Ed., W.H.Freeman & Co., New York, 1997.

Typically, a naturally occurring immunoglobulin has heavy (H) chains andlight (L) chains interconnected by disulfide bonds. There are two typesof light chain, lambda (λ) and kappa (k). There are five main heavychain classes (or isotypes) which determine the functional activity ofan antibody molecule: IgM, IgD, IgG, IgA and IgE.

Each heavy and light chain contains a constant region and a variableregion, (the regions are also known as “domains”). In combination, theheavy and the light chain variable regions specifically bind theantigen. Light and heavy chain variable regions contain a “framework”region interrupted by three hypervariable regions, also called“complementarity-determining regions” or “CDRs”. The extent of theframework region and CDRs have been defined (see, Kabat et al.,Sequences of Proteins of Immunological Interest, U.S. Department ofHealth and Human Services, 1991). The Kabat database is now maintainedonline. The sequences of the framework regions of different light orheavy chains are relatively conserved within a species. The frameworkregion of an antibody, that is the combined framework regions of theconstituent light and heavy chains, serves to position and align theCDRs in three-dimensional space.

The CDRs are primarily responsible for binding to an epitope of anantigen. The CDRs of each chain are typically referred to as CDR1, CDR2,and CDR3, numbered sequentially starting from the N-terminus, and arealso typically identified by the chain in which the particular CDR islocated. Thus, a V_(H) CDR3 is located in the variable domain of theheavy chain of the antibody in which it is found, whereas a V_(L) CDR1is the CDR1 from the variable domain of the light chain of the antibodyin which it is found. An antibody that binds RET will have a specificV_(H) region and the V_(L) region sequence, and thus specific CDRsequences. Antibodies with different specificities (such as differentcombining sites for different antigens) have different CDRs. Although itis the CDRs that vary from antibody to antibody, only a limited numberof amino acid positions within the CDRs are directly involved in antigenbinding. These positions within the CDRs are called specificitydetermining residues (SDRs).

References to “V_(H)” or “VH” refer to the variable region of animmunoglobulin heavy chain, including that of an Fv, scFv, dsFv or Fab.References to “V_(L)” or “VL” refer to the variable region of animmunoglobulin light chain, including that of an Fv, scFv, dsFv or Fab.

A “monoclonal antibody” is an antibody produced by a single clone ofB-lymphocytes or by a cell into which the light and heavy chain genes ofa single antibody have been transfected. Monoclonal antibodies areproduced by methods known to those of skill in the art, for instance bymaking hybrid antibody-forming cells from a fusion of myeloma cells withimmune spleen cells. Monoclonal antibodies include humanized monoclonalantibodies.

A “polyclonal antibody” is an antibody that is derived from differentB-cell lines. Polyclonal antibodies are a mixture of immunoglobulinmolecules secreted against a specific antigen, each recognizing adifferent epitope. These antibodies are produced by methods known tothose of skill in the art, for instance, by injection of an antigen intoa suitable mammal (such as a mouse, rabbit or goat) that induces theB-lymphocytes to produce IgG immunoglobulins specific for the antigen,which are then purified from the mammal's serum.

A “chimeric antibody” has framework residues from one species, such ashuman, and CDRs (which generally confer antigen binding) from anotherspecies, such as a murine antibody that specifically binds a musculardystrophy-associated molecule.

A “humanized” immunoglobulin is an immunoglobulin including a humanframework region and one or more CDRs from a non-human (for example amouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulinproviding the CDRs is termed a “donor,” and the human immunoglobulinproviding the framework is termed an “acceptor.” In one example, all theCDRs are from the donor immunoglobulin in a humanized immunoglobulin.Constant regions need not be present, but if they are, they are lyidentical to human immunoglobulin constant regions, e.g., at least about85-90%, such as about 95% or more identical. Hence, all parts of ahumanized immunoglobulin, except possibly the CDRs, are substantiallyidentical to corresponding parts of natural human immunoglobulinsequences. Humanized immunoglobulins can be constructed by means ofgenetic engineering (see for example, U.S. Pat. No. 5,585,089).

Alteration or modulation in expression: An alteration in expression of agene, gene product or modulator thereof, such as one or more musculardystrophy associated molecules disclosed herein, refers to a change ordifference, such as an increase or decrease, in the level of the gene,gene product, or modulators thereof that is detectable in a biologicalsample (such as a sample from a subject at risk or having musculardystrophy) relative to a control (such as a sample from a subjectwithout a muscular dystrophy) or a reference value known to beindicative of the level of the gene, gene product or modulator thereofin the absence of the disease. An “alteration” in expression includes anincrease in expression (up-regulation) or a decrease in expression(down-regulation).

Analog: A compound which is sufficiently homologous to a compound suchthat it has a similar functional activity for a desired purpose as theoriginal compound. Analogs include polypeptides having one or more aminoacid substitutions compared with a particular substance.

At least substantially homologous: A phrase used in the presentdisclosure, refers to a degree of homology sufficient to produce atleast a portion of the activity of a reference material in muscleregeneration, maintenance or repair, or wound healing. In some examples,materials are at least substantially homologous when they are at leastabout 95%, at least about 98%, or at least about 99% homologous to areference material.

Biological activity: The beneficial or adverse effects of an agent onliving matter. When the agent is a complex chemical mixture, thisactivity is exerted by the substance's active ingredient orpharmacophore, but can be modified by the other constituents. Activityis generally dosage-dependent and it is not uncommon to have effectsranging from beneficial to adverse for one substance when going from lowto high doses. In one example, the agent significantly reduces thebiological activity of the one or more muscular dystrophy associatedmolecules disclosed herein which reduces one or more signs or symptomsassociated with the muscular dystrophy.

Biomarkers: Natural substances produced by the body that are used asindicators of specific biological states. Biomarkers allow conditions,including diseases to be diagnosed, progression of such monitored, aswell as to test the efficacy of disease treatments. The musculardystrophies are one group of diseases with a lack of biomarkers. Serumcreatine kinase (a byproduct of muscle breakdown) levels have previouslybeen used as a biomarker for muscular dystrophy but do not accuratelyfollow the progression of the disease. Disclosed herein are biomarkersfor muscular dystrophy. In particular examples, the biomarker indicatesa particular type of muscular dystrophy to be present or the severity ofthe condition (e.g., an increase in the level of Galectin-3 indicates apoor prognosis).

Bone density: A term referring to the amount of mineral matter persquare centimeter of bones. Bone density is used in clinical medicine asan indirect indicator for various conditions and diseases, including,but not limited to, osteoporosis and fracture risk. Many techniques areavailable to determine bone density, but ultrasound has been describedas a more cost-effective approach. The test works by measuring aspecific bone or bones, usually the spine, hip, and wrist. The densityof these bones is then compared with an average index based on age, sex,and size. The resulting comparison is used to determine risk forfractures and the stage of osteoporosis in an individual. Average bonemineral density=BMC/W [g/cm2]; BMC=bone mineral content=g/cm; andW=width at the scanned line. Results are generally scored by twomeasures, the T-score and the Z-score. Scores indicate the amount one'sbone mineral density varies from the mean. Negative scores indicatelower bone density, and positive scores indicate higher.

The T-score is the relevant measure when screening for osteoporosis. Itis the bone mineral density at the site when compared to the youngnormal reference mean. It is a comparison of a patient's BMD to that ofa healthy thirty-year-old. The US standard is to use data for athirty-year-old of the same sex and ethnicity, but the WHO recommendsusing data for a thirty-year-old white female for everyone. Values forthirty-year-olds are used in post-menopausal women and men over age 50because they better predict risk of future fracture. The criteria of theWorld Health Organization are: Normal is a T-score of −1.0 or higher;Osteopenia is defined as between −1.0 and −2.5; and Osteoporosis isdefined as −2.5 or lower, meaning a bone density that is two and a halfstandard deviations below the mean of a thirty-year-old man/woman.

The Z-score is the comparison to the age-matched normal and is usuallyused in cases of severe osteoporosis. This is the number of standarddeviations a subject's BMD differs from the average BMD of their age,sex, and ethnicity. This value is used in premenopausal women, men underthe age of 50, and in children. It is most useful when the score is lessthan 2 standard deviations below this normal. In this setting, it ishelpful to scrutinize for coexisting illnesses that may contribute toosteoporosis such as glucocorticoid therapy, hyperparathyroidism, oralcoholism.

Contacting: Placement in direct physical association, including both asolid and liquid form. Contacting an agent with a cell can occur invitro by adding the agent to isolated cells or in vivo by administeringthe agent to a subject.

Control: A sample or standard used for comparison with a test sample,such as a biological sample obtained from a patient (or plurality ofpatients) without a particular disease or condition, such as a musculardystrophy. In some embodiments, the control is a sample obtained from ahealthy patient (or plurality of patients) (also referred to herein as a“normal” control), such as a normal biological sample. In someembodiments, the control is a historical control or standard value(e.g., a previously tested control sample or group of samples thatrepresent baseline or normal values (e.g., expression values), such asbaseline or normal values of a particular gene, gene product in asubject without a muscular dystrophy). In some examples, the control isa standard value representing the average value (or average range ofvalues) obtained from a plurality of patient samples (such as an averagevalue or range of values of the gene or gene products in the subjectswithout a muscular dystrophy).

Decrease: To reduce the quality, amount, or strength of something. Inone example, a therapy decreases one or more symptoms associated withthe muscular dystrophy, for example as compared to the response in theabsence of the therapy. In a particular example, a therapy decreases(also known as down-regulates) the expression of a musculardystrophy-associated molecule, such as a decrease of at least 10%, atleast 20%, at least 50%, or even at least 90%, thereby increasing asubject's chance of survival. In some examples, a decrease in expressionrefers to any process which results in a decrease in production of oneor more molecules associated with muscular dystrophy. A gene product canbe RNA (such as mRNA, rRNA, tRNA, and structural RNA) or protein,Therefore, gene downregulation or deactivation includes processes thatdecrease transcription of a gene or translation of mRNA.

Examples of processes that decrease transcription include those thatfacilitate degradation of a transcription initiation complex, those thatdecrease transcription initiation rate, those that decreasetranscription elongation rate, those that decrease processivity oftranscription and those that increase transcriptional repression. Genedownregulation can include reduction of expression above an existinglevel. Examples of processes that decrease translation include thosethat decrease translational initiation, those that decreasetranslational elongation and those that decrease mRNA stability.

Gene downregulation includes any detectable decrease in the productionof a gene product. In certain examples, production of a gene productdecreases by at least 2-fold, for example at least 3-fold or at least4-fold, as compared to a control (such an amount of gene expression in anormal cell). In one example, a control is a relative amount of geneexpression or protein expression in a biological sample taken from asubject who does not have muscular dystrophy, such as DMD or MDC1A. Suchdecreases can be measured using the methods disclosed herein. Forexample, “detecting or measuring expression of a gene product” includesquantifying the amount of the gene, gene product or modulator thereofpresent in a sample. Quantification can be either numerical or relative.Detecting expression of the gene, gene product or modulators thereof canbe achieved using any method known in the art or described herein, suchas by measuring nucleic acids by PCR (such as RT-PCR) and proteins byELISA. In primary embodiments, the change detected is an increase ordecrease in expression as compared to a control, such as a referencevalue or a healthy control subject. In some examples, the detectedincrease or decrease is an increase or decrease of at least two-foldcompared with the control or standard. Controls or standards forcomparison to a sample, for the determination of differentialexpression, include samples believed to be normal (in that they are notaltered for the desired characteristic, for example a sample from asubject who does not have muscular dystrophy, such as DMD or MDC1A) aswell as laboratory values (e.g., range of values), even though possiblyarbitrarily set, keeping in mind that such values can vary fromlaboratory to laboratory.

Laboratory standards and values can be set based on a known ordetermined population value and can be supplied in the format of a graphor table that permits comparison of measured, experimentally determinedvalues.

The level of expression in either a qualitative or quantitative mannercan detect nucleic acid or protein. Exemplary methods include microarrayanalysis, RT-PCR, Northern blot, Western blot, and mass spectrometry.

Derivative: A form of a substance, such as a galectin or portionthereof, which has at least one functional group altered, added, orremoved, compared with the parent compound.

Diagnosis: The process of identifying a disease, such as musculardystrophy, by its signs, symptoms and results of various tests. Theconclusion reached through that process is also called “a diagnosis.”Forms of testing commonly performed include blood tests, medicalimaging, urinalysis, and biopsy.

Effective amount: An amount of agent that is sufficient to generate adesired response, such as reducing or inhibiting one or more signs orsymptoms associated with a condition or disease. When administered to asubject, a dosage will generally be used that will achieve targettissue/cell concentrations. In some examples, an “effective amount” isone that treats one or more symptoms and/or underlying causes of any ofa disorder or disease. In some examples, an “effective amount” is atherapeutically effective amount in which the agent alone with anadditional therapeutic agent(s) (for example anti-pathogenic agents),induces the desired response such as treatment of a muscular dystrophy,such as DMD, LGMD, FHMD, Beckers muscular dystrophy or MDC1A.

In particular examples, it is an amount of an agent capable ofmodulating one or more of the disclosed genes, gene products ormodulators thereof associated with a muscular dystrophy by least 20%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 98%, or even at least 100% (elimination of thedisease to a point beyond detection) by the agent.

In some examples, an effective amount is an amount of a pharmaceuticalpreparation that alone, or together with a pharmaceutically acceptablecarrier or one or more additional therapeutic agents, induces thedesired response.

In one example, a desired response is to increase the subject's survivaltime by slowing the progression of the disease. The disease does notneed to be completely inhibited for the pharmaceutical preparation to beeffective. For example, a pharmaceutical preparation can decrease theprogression of the disease by a desired amount, for example by at least20%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 95%, at least 98%, or even at least 100%, as compared tothe progression typical in the absence of the pharmaceuticalpreparation.

In another or additional example, it is an amount sufficient topartially or completely alleviate symptoms of the muscular dystrophywithin the subject. Treatment can involve only slowing the progressionof the disease temporarily, but can also include halting or reversingthe progression of the disease permanently.

Effective amounts of the agents described herein can be determined inmany different ways, such as assaying for a reduction in of one or moresigns or symptoms associated with the muscular dystrophy in the subjector measuring the expression level of one or more molecules known to beassociated with the muscular dystrophy. Effective amounts also can bedetermined through various in vitro, in vivo or in situ assays,including the assays described herein.

The disclosed therapeutic agents can be administered in a single dose,or in several doses, for example daily, during a course of treatment.However, the effective amount can be dependent on the source applied(for example a nucleic acid molecule isolated from a cellular extractversus a chemically synthesized and purified nucleic acid), the subjectbeing treated, the severity and type of the condition being treated, andthe manner of administration.

Expression: The process by which the coded information of a gene isconverted into an operational, non-operational, or structural part of acell, such as the synthesis of a protein. Gene expression can beinfluenced by external signals. For instance, exposure of a cell to ahormone may stimulate expression of a hormone-induced gene. Differenttypes of cells can respond differently to an identical signal.Expression of a gene also can be regulated anywhere in the pathway fromDNA to RNA to protein. Regulation can include controls on transcription,translation, RNA transport and processing, degradation of intermediarymolecules such as mRNA, or through activation, inactivation,compartmentalization or degradation of specific protein molecules afterthey are produced. In an example, gene expression can be monitored todiagnosis and/or prognosis a subject with muscular dystrophy, such aspredict a subject's survival time with DMD, LGMD or MDC1A.

The expression of a nucleic acid molecule can be altered relative to anormal (wild type) nucleic acid molecule. Alterations in geneexpression, such as differential expression, include but are not limitedto: (1) overexpression; (2) underexpression; or (3) suppression ofexpression. Alternations in the expression of a nucleic acid moleculecan be associated with, and in fact cause, a change in expression of thecorresponding protein.

Protein expression can also be altered in some manner to be differentfrom the expression of the protein in a normal (wild type) situation.This includes but is not necessarily limited to: (1) a mutation in theprotein such that one or more of the amino acid residues is different;(2) a short deletion or addition of one or a few (such as no more than10-20) amino acid residues to the sequence of the protein; (3) a longerdeletion or addition of amino acid residues (such as at least 20residues), such that an entire protein domain or sub-domain is removedor added; (4) expression of an increased amount of the protein comparedto a control or standard amount; (5) expression of a decreased amount ofthe protein compared to a control or standard amount; (6) alteration ofthe subcellular localization or targeting of the protein; (7) alterationof the temporally regulated expression of the protein (such that theprotein is expressed when it normally would not be, or alternatively isnot expressed when it normally would be); (8) alteration in stability ofa protein through increased longevity in the time that the proteinremains localized in a cell; and (9) alteration of the localized (suchas organ or tissue specific or subcellular localization) expression ofthe protein (such that the protein is not expressed where it wouldnormally be expressed or is expressed where it normally would not beexpressed), each compared to a control or standard. Controls orstandards for comparison to a sample, for the determination ofdifferential expression, include samples believed to be normal (in thatthey are not altered for the desired characteristic, for example asample from a subject who does not have muscular dystrophy, such as DMDor MDC1A) as well as laboratory values (e.g., range of values), eventhough possibly arbitrarily set, keeping in mind that such values canvary from laboratory to laboratory.

Laboratory standards and values can be set based on a known ordetermined population value and can be supplied in the format of a graphor table that permits comparison of measured, experimentally determinedvalues.

Extracellular matrix: The extracellular structure of a tissue or a layerthereof, including the arrangement, composition, and forms of one ormore matrix components, such as proteins, including structural proteinssuch as collagen and elastin, proteins such as fibronectin and laminins,and proteoglycans. The matrix may comprise fibrillic collagen, having anetwork of fibers. In some examples, the extracellular matrix isconnected to cells through the costameric protein network.

Fragment: A portion of a substance, such as galectin. A fragment may be,in some examples, a particular domain or chain of a protein. Forexample, particular embodiments of the present disclosure involveadministering a fragment of galectin, such as a fragment of Galectin-1or Galectin-3. Fragments may be synthetic or may be derived from largerparent substances.

Functional group: A radical, other than a hydrocarbon radical, that addsa physical or chemical property to a substance.

Galectins: β-galactoside-binding animal lectins that modulateextracellular matrix interactions, cell attachment and differentiation,as well as cancer invasion and metastasis, Fifteen mammalian galectinshave been identified thus far, with Galectin-1 and Galectin-3 being twoof the most well characterized. Galectin-1 is encoded by the Lgals1gene, located on chromosome 22q12. Galectin-1, approximately 15 kDa insize, binds to a number of extracellular matrix components, such aslaminin, as well as with several integrins, including the α7β1 integrin.It is present both intracellularly and extracellularly and has beenshown to play a role in immunosupression, cell-growth regulation, cellapoptosis and pre-mRNA slicing. Galectin-1 is found in skeletal muscleand has been implicated in the conversion of dermal fibroblasts tomuscle due to its competition with laminin for α7β1 integrin binding. .

Galectin-3, about 30 kDa in size, is encoded by the Lgals3 gene, locatedon chromosome 14q22. This protein has a carboxyl-terminal domain thatbinds carbohydrates and an amino terminal domain that cross-linkscarbohydrate and noncarbohydrate ligands. Similar to Galectin-1,Galectin-3 is also found both intracellularly and extracellularly.Intracellularly, Galectin-3 has been shown to regulate the cell cycleand apoptosis. Extracellularly, Galectin-3 works to mediate cell-cellinteractions as well as cell-extracellular matrix interactions.Galectin-3 is also expressed and secreted by macrophages and monocytes.Galectin-3 is specifically upregulated during monocyte differentiation,and downregulated during differentiation into dendritic cells.

In some examples, expression of Galectin-1 is increased in a subjectwith muscular dystrophy, such as with DMD, MDC1A, FHMD, Beckers musculardystrophy or LGMD. The term Galectin-1 includes any Galectin-1 gene,cDNA, mRNA, or protein from any organism and that is Galectin-1 and isexpressed in a sample from a subject with muscular dystrophy such asDMD, LGMD, FHMD, Beckers muscular dystrophy or MDC1A.

In some examples, expression of Galectin-3 is increased in a subjectwith muscular dystrophy, such as with DMD, MDC1A, FHMD, Beckers musculardystrophy or LGMD. The term Galectin-3 includes any Galectin-3 gene,cDNA, mRNA, or protein from any organism and that is Galectin-3 and isexpressed in a sample from a subject with muscular dystrophy such asDMD, LGMD, FHMD, Beckers muscular dystrophy or MDC1A.

Nucleic acid and protein sequences for Galectin-1 and Galectin-3 arepublicly available. For example, GENBANK® Accession Nos: NM_(—)002306;NM_(—)003225; NM_(—)00177388 disclose Galectin-1 nucleic acid sequences,and GENBANK® Accession No.: NP_(—)002296 discloses a Galectin-1 proteinsequence, all of which are incorporated by reference as provided byGENBANK® on Aug. 11, 2011; GENBANK® Accession No. NP_(—)032521.1 alsoprovides a Galectin-1 protein sequence which is incorporated byreference in its entirety as provided by GENBANK® on Aug. 10, 2012.

GENBANK® Accession Nos: NM_(—)001177388; NM_(—)002306; NP_(—)003225disclose Galectin-3 nucleic acid sequences, and GENBANK® AccessionNos.:_BA22164 discloses a Galectin-3 protein sequence, all of which areincorporated by reference as provided by GENBANK® on Aug. 11, 2011;GENBANK® Accession No. NP_(—)034835.1 also provides a Galectin-3 proteinsequence which is incorporated by reference in its entirety as providedby GENBANK® on Aug. 10, 2012.

In one example, Galectin-1 includes a full-length wild-type (or native)sequence, as well as Galectin-1 allelic variants, fragments, homologs orfusion sequences, such as Galectin-1 allelic variants, fragments,homologs or fusion sequences that retain the ability to increase α7integrin expression or biological activity. In certain examples,Galectin-1 has at least 80% sequence identity, for example at least 85%,90%, 95%, or 98% sequence identity to Galectin-1.

In one example, Galectin-3 includes a full-length wild-type (or native)sequence, as well as Galectin-3 allelic variants, fragments, homologs orfusion sequences, such as Galectin-3 allelic variants, fragments,homologs or fusion sequences that retain the ability to increase α7integrin expression or biological activity. In certain examples,Galectin-3 has at least 80% sequence identity, for example at least 85%,90%, 95%, or 98% sequence identity to Galectin-3.

Improving muscular health: An improvement in muscular health comparedwith a preexisting state or compared with a state which would occur inthe absence of treatment. For example, improving muscular health mayinclude enhancing muscle regeneration, maintenance, or repair. Improvingmuscular health may also include prospectively treating a subject toprevent or reduce muscular damage or injury.

Inhibiting a disease or condition: A phrase referring to inhibiting thedevelopment of a disease or condition, such as reducing, decreasing ordelaying a sign or symptom associated with the disease or condition, forexample, in a subject who is at risk of acquiring the disease/conditionor has the particular disease/condition. Particular methods of thepresent disclosure provide methods for inhibiting muscular dystrophy.

Kyphosis: A condition of over-curvature of the thoracic vertebrae (upperback). It can be either the result of degenerative diseases (such asarthritis), developmental problems (the most common example beingScheuermann's disease), osteoporosis with compression fractures of thevertebrae, or trauma. In the sense of a deformity, it is thepathological curving of the spine, where parts of the spinal column losesome or all of their lordotic profile. This causes a bowing of the back,seen as a slouching posture. The Cobb angle is the preferred method ofmeasuring kyphosis.

Label: An agent capable of detection, for example by ELISA,spectrophotometry, flow cytometry, or microscopy. For example, a labelcan be attached to a nucleic acid molecule or protein (such asGalectin-1 or Galectin-3), thereby permitting detection of the nucleicacid molecule or protein. Examples of labels include, but are notlimited to, radioactive isotopes, enzyme substrates, co-factors,ligands, chemiluminescent agents, fluorophores, haptens, enzymes, andcombinations thereof. Methods for labeling and guidance in the choice oflabels appropriate for various purposes are discussed for example inSambrook et al. (Molecular Cloning: A Laboratory Manual, Cold SpringHarbor, New York, 1989) and Ausubel et al. (In Current Protocols inMolecular Biology, John Wiley & Sons, New York, 1998). In a particularexample, a label is conjugated to an agent that binds to one or more ofthe muscular dystrophy associated molecules, such as Galectin-1 orGalectin-3, to allow for the detection and prognosis of the disease in asubject.

Lupus erythematosus: A term referring to a collection of autoimmunediseases, in which the human immune system becomes hyperactive andattacks normal, healthy tissues. Symptoms of these diseases can affectmany different body systems, including joints, skin, kidneys, bloodcells, heart, and lungs.Maintenance of cells or tissue: A phrase refersto maintaining cells or tissue, such as muscle cells or muscle tissue,in at least substantially the same physiological condition, such asmaintaining such condition even in the presence of stimulus which wouldnormally cause damage, injury, or disease.

Multiple sclerosis: An autoimmune disease classically described as acentral nervous system white matter disorder disseminated in time andspace that presents as relapsing-remitting illness in 80-85% ofpatients, Diagnosis can be made by brain and spinal cord magneticresonance imaging (MRI), analysis of somatosensory evoked potentials,and analysis of cerebrospinal fluid to detect increased amounts ofimmunoglobulin or oligoclonal bands. MRI is a particularly sensitivediagnostic tool. MRI abnormalities indicating the presence orprogression of MS include hyperintense white matter signals onT2-weighted and fluid attenuated inversion recovery images, gadoliniumenhancement of active lesions, hypointensive “black holes” (representinggliosis and axonal pathology), and brain atrophy on T1-weighted studies.Serial MRI studies can be used to indicate disease progression.Relapsing-remitting multiple sclerosis is a clinical course of MS thatis characterized by clearly defined, acute attacks with full or partialrecovery and no disease progression between attacks.Secondary-progressive multiple sclerosis is a clinical course of MS thatinitially is relapsing-remitting, and then becomes progressive at avariable rate, possibly with an occasional relapse and minor remission.Primary progressive multiple sclerosis presents initially in theprogressive form.

Muscle: Any myoblast, myocyte, myofiber, myotube or other structurecomposed of muscle cells. Muscles or myocytes can be skeletal, smooth,or cardiac. Muscle may also refer to, in particular implementations ofthe present disclosure, cells or other materials capable of formingmyocytes, such as stem cells and satellite cells.

Muscle density: A term referring to the rigidity (“hardness”) of amuscle during a resting state. Muscular density is also referred to asmuscle tone (e.g., the continuous and passive partial contraction of themuscles, or the muscle's resistance to passive stretch during restingstate).

Muscle strength: The amount of force a muscle can produce with a singlemaximal effort.

Muscular dystrophy: A term used to refer to a group of genetic disordersthat lead to progressive muscle weakness. Muscular dystrophy can resultin skeletal muscle weakness and defects in skeletal muscle proteins,leading to a variety of impaired physiological functions. Nosatisfactory treatment of muscular dystrophy exists. Existing treatmentstypically focus on ameliorating the effects of the disease and improvingthe patient's quality of life, such as through physical therapy orthrough the provision of orthopedic devices.

Mutated genes associated with muscular dystrophy are responsible forencoding a number of proteins associated with the costameric proteinnetwork. Such proteins include laminin-2, collagen, dystroglycan,integrins, caveolin-3, ankyrin, dystrophin, a-dystrobrevin, vinculin,plectin, BPAG1b, muscle LIM protein, desmin, actinin-associated LIMprotein, a-actin, titin, telethonin, cypher, myotilin, and thesarcoglycan/sarcospan complex.

The most common form of muscular dystrophy is Duchenne musculardystrophy (DMD), affecting 1 in 3,500 live male births. DMD is anX-linked recessive disorder characterized by a mutation in the gene thatcodes for dystrophin. Dystrophin is a cytoskeletal protein about 430 kDain size. This protein works to connect the cell's cytoskeleton andextracellular matrix. The loss of dystrophin in DMD patients leads to aloss of muscle fiber attachment at the extracellular matrix duringcontraction, which ultimately leads to progressive fiber damage,membrane leakage and a loss of muscle function. Most patients die beforethey reach the age of 30 due to respiratory or cardiac failure.

Beckers muscular dystrophy (also known as Benign pseudohypertrophicmuscular dystrophy) is related to Duchenne muscular dystrophy in thatboth result from a mutation in the dystrophin gene, but in Duchennemuscular dystrophy no functional dystrophin is produced making DMD muchmore severe than BMD. BMD is an X-linked recessive inherited disordercharacterized by slowly progressive muscle weakness of the legs andpelvis. BMD is a type of dystrophinopathy, which includes a spectrum ofmuscle diseases in which there is insufficient dystrophin produced inthe muscle cells, results in instability in the structure of muscle cellmembrane. This is caused by mutations in the dystrophin gene, whichencodes the protein dystrophin. The pattern of symptom development ofBMD is similar to DMD, but with a later, and much slower rate ofprogression.

Congenital muscular dystrophies are caused by gene mutations affectingthe production of other costameric proteins. MDC1A is a congentialmuscular dystrophy due to a genetic mutation in the LAMA2 gene whichresults in lack of or complete loss of laminin-α2 protein. This loss oflaminin-α2 leads to an absence of laminins-211/221. Laminins-211/221 aremajor components of the extracellular matrix and play a key role inmuscle cell development. During muscle cell differentiation lamininbinds to the α7β1 integrin. Without laminin-α2, muscle fibers are unableto adhere to the basement membrane and myotubes undergo apotosis. Muscleregeneration also fails, leading to a loss of muscle repair and anincrease in muscle fibrosis and inflammation. This chronic tissue injuryis a major cause of morbidity and mortality in MDC1A.

Congenital Muscular Dystrophies (CMD) and Limb-Girdle muscular dystrophy(LGMD) are common forms of highly heterogeneous muscular dystrophieswhich can be distinguished by their age at onset. In CMD, onset ofsymptoms is at birth or within the first 6 months of life; in LGMD onsetof symptoms is in late childhood, adolescence or even adult life.Inheritance in LGMD can be autosomal dominant (LGMD type 1) or autosomalrecessive (LGMD type 2), CMD is recessively inherited. CMD and LGMD canoverlap both clinically and genetically

MDC1A is a progressive muscle wasting disease that results in childrenbeing confined to a wheelchair, requiring ventilator assistance tobreathe and premature death. Symptoms are detected at birth with poormuscle tone and “floppy” baby syndrome. DMD, BMD and LGMD areprogressive muscle degenerative diseases usually diagnosed at 3-5 yearsof age when children show developmental delay including ability to walkand climb stairs. The disease is progressive and children are usuallyconfined to a wheelchair in their teens and require ventilatorassistance.

Facioscapulohumeral muscular dystrophy (FHMD) is a form of musculardystrophy associated with progressive muscle weakness and loss of muscletissue. Unlike DMD and BMD which mainly affect the lower body, FHMDaffects the upper body mainly the face, shoulder and upper arm muscles.However, it can affect muscles around the pelvis, hips, and lower leg.Symptoms for FHMD often do not appear until age 10-26, but it is notuncommon for symptoms to appear much later. In some cases, symptomsnever develop. Symptoms are usually mild and very slowly become worse.Facial muscle weakness is common, and may include eyelid drooping,inability to whistle, decreased facial expression, depressed or angryfacial expression, difficulty pronouncing words, shoulder muscleweakness (leading to deformities such as pronounced shoulder blades(scapular winging) and sloping shoulders), weakness of the lower,hearing loss and possible heart conditions.

Muscular dystrophy-associated molecule: A molecule whose expression orbiological activity is altered in subject with muscular dystrophy. Suchmolecules include, for instance, nucleic acid sequences (such as DNA,cDNA, or mRNAs) and proteins. Specific genes include those disclosedherein, including the Examples, as well as fragments of the full-lengthgenes, cDNAs, or mRNAs (and proteins encoded thereby) whose expressionis altered (such as upregulated or downregulated) in response tomuscular dystrophy, including DMD, LGMD, FHMD, Beckers musculardystrophy and/or MDC1A. Thus, the presence or absence of the respectivemuscular dystrophy-associated molecules can be used to diagnose and/ordetermine the prognosis of a muscular dystrophy, and in particular DMD,LGMD, FHMD, Beckers muscular dystrophy or MDC1A in a subject as well asto treat a subject with a muscular dystrophy, such as DMD, LGMD, FHMD,Beckers muscular dystrophy or MDC1A. In some examples, it is a moleculeassociated with one or more signs or symptoms of a musculardystrophy-associated condition or disease. In some examples, a musculardystrophy-associated molecule is one or more molecules associated withDMD, LGMD, FHMD, Beckers muscular dystrophy and/or MDC1A, such asGalectin-1 or Galectin-3.

Osteoporosis: A progressive bone disease that's characterized by adecrease in bone mass and density and that leads to an increased risk offracture. In osteoporosis, the bone mineral density (BMD) is reduced,bone microarchitecture deteriorates, and the amount and variety ofproteins in bone are altered. Osteoporosis is defined by the WorldHealth Organization (WHO) as a bone mineral density of 2.5 standarddeviations or more below the mean peak bone mass (average of young,healthy adults) as measured by dual-energy X-ray absorptiometry; theterm “established osteoporosis” includes the presence of a fragilityfracture. The disease may be classified as primary type 1, primary type2, or secondary.

The form of osteoporosis most common in women after menopause isreferred to as primary type 1 or postmenopausal osteoporosis. Primarytype 2 osteoporosis or senile osteoporosis occurs after age 75 and isseen in both females and males at a ratio of 2:1. Secondary osteoporosismay arise at any age and affect men and women equally. This form resultsfrom chronic predisposing medical problems or disease, or prolonged useof medications such as glucocorticoids, when the disease is calledsteroid- or glucocorticoid-induced osteoporosis.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers (vehicles) useful in this disclosure are conventional.Remington's Pharmaceutical Sciences, by E. W. Martin, Mack PublishingCo., Easton, Pa., 19th Edition (1995), describes compositions andformulations suitable for pharmaceutical delivery of one or moretherapeutic agents, such as one or more compositions that include abinding agent that specifically binds to at least one of the disclosedmuscular dystrophy-associated molecules.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationscan include injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. In addition to biologically-neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate, sodium lactate, potassium chloride,calcium chloride, and triethanolamine oleate.

Polymerase Chain Reaction (PCR): An in vitro amplification techniquethat increases the number of copies of a nucleic acid molecule (forexample, a nucleic acid molecule in a sample or specimen). In anexample, a biological sample collected from a subject is contacted witha pair of oligonucleotide primers, under conditions that allow for thehybridization of the primers to nucleic acid template in the sample(such as those listed in Example 1 or 2). The primers are extended undersuitable conditions, dissociated from the template, and thenre-annealed, extended, and dissociated to amplify the number of copiesof the nucleic acid. The product of a PCR can be characterized byelectrophoresis, restriction endonuclease cleavage patterns,oligonucleotide hybridization or ligation, and/or nucleic acidsequencing, using standard techniques or other standard techniques knownin the art.

Prognosis: A prediction of the course of a disease, such as musculardystrophy. The prediction can include determining the likelihood of asubject to develop aggressive, recurrent disease, to survive aparticular amount of time (e.g. determine the likelihood that a subjectwill survive 1, 2, 3 or 5 years), to respond to a particular therapy orcombinations thereof.

Regeneration: The repair of cells or tissue, such as muscle cells ortissue (or organs) which includes muscle cells, following injury ordamage to at least partially restore the muscle or tissue to a conditionsimilar to which the cells or tissue existed before the injury or damageoccurred. Regeneration also refers to facilitating repair of cells ortissue in a subject having a disease affecting such cells or tissue toeliminate or ameliorate the effects of the disease. In more specificexamples, regeneration places the cells or tissue in the same conditionor an improved physiological condition as before the injury or damageoccurred or the condition which would exist in the absence of disease.

Repair of cells or tissue: A phrase which refers to the physiologicalprocess of healing damage to the cells or tissue such as muscle cells ortissue (or organs) following damage or other trauma.

Rheumatoid arthritis (RA): An autoimmune disease that results in achronic, systemic inflammatory disorder that may affect many tissues andorgans, but principally attacks flexible (synovial) joints. It can be adisabling and painful condition, which can lead to substantial loss offunctioning and mobility if not adequately treated. The process involvesan inflammatory response of the capsule around the joints (synovium)secondary to swelling (turgescence) of synovial cells, excess synovialfluid, and the development of fibrous tissue (pannus) in the synovium.The pathology of the disease process often leads to the destruction ofarticular cartilage and ankylosis (fusion) of the joints. RA can alsoproduce diffuse inflammation in the lungs, the membrane around the heart(pericardium), the membranes of the lung (pleura), and white of the eye(sclera), and also nodular lesions, most common in subcutaneous tissue.RA is a clinical diagnosis made on the basis of symptoms, physical exam,radiographs (X-rays) and laboratory tests.

Sample (or biological sample): A biological specimen containing genomicDNA, RNA (including mRNA), protein, or combinations thereof, obtainedfrom a subject. Examples include, but are not limited to, peripheralblood, urine, saliva, tissue biopsy, surgical specimen, and autopsymaterial. In one example, a sample includes muscle biopsy, such as froma subject with DMD, LGMD, FHMD, Beckers muscular dystrophy or MDC1A.

Scoliosis: A condition in which a person's spine is curved from side toside. Although it is a complex three-dimensional deformity, on an X-ray,viewed from the rear, the spine of an individual with scoliosis canresemble an “S” or a “C”, rather than a straight line. Scoliosis istypically classified as either congenital (caused by vertebral anomaliespresent at birth), idiopathic (cause unknown, subclassified asinfantile, juvenile, adolescent, or adult, according to when onsetoccurred), or secondary to a primary condition. Scoliosis is defined asa spinal curvature of more than 10 degrees to the right or left as theexaminer faces the subject (in the coronal plane). Deformity may alsoexist to the front or back (in the sagittal plane). The standard methodfor assessing the curvature quantitatively is measurement of the Cobbangle, which is the angle between two lines, drawn perpendicular to theupper endplate of the uppermost vertebra involved and the lower endplateof the lowest vertebra involved.

Signs or symptoms: Any subjective evidence of disease or of a subject'scondition, e.g., such evidence as perceived by the subject; a noticeablechange in a subject's condition indicative of some bodily or mentalstate, A “sign” is any abnormality indicative of disease, discoverableon examination or assessment of a subject. A sign is generally anobjective indication of disease. Signs include, but are not limited toany measurable parameters such as tests for detecting musculardystrophy, including measuring creatine kinase levels, electromyography(to determine if weakness is caused by destruction of muscle tissuerather than by damage to nerves) orimmunohistochemistry/immunoblotting/immunoassay (e.g., ELISA) to measuremuscular dystrophy-associated molecules. In one example, reducing orinhibiting one or more symptoms or signs associated with musculardystrophy, includes reducing or inhibiting the activity or expression ofone or more disclosed muscular dystrophy-associated molecules by adesired amount, for example by at least 20%, at least 50%, at least 60%,at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, oreven at least 100%, as compared to the activity and/or expression in theabsence of the treatment. Symptoms of muscular dystrophy include, butare not limited to, muscle weakness and loss, difficulty running,difficulty hopping, difficulty jumping, difficulty walking, difficultybreathing, fatigue, skeletal deformities, muscle deformities(contractions of heels; pseudohypertrophy of calf muscles), heartdisease (such as dilated cardiomyopathy), elevated creatinephosphokinase (CK) levels in blood or combinations thereof,

Subject: Living multi-cellular vertebrate organisms, a category thatincludes human and non-human mammals.

Therapeutically-effective amount: An amount effective for lessening,ameliorating, eliminating, preventing, or inhibiting at least onesymptom of a disease, disorder, or condition treated and may beempirically determined. In various embodiments of the presentdisclosure, a “therapeutically-effective amount” is a “muscleregeneration promoting-amount,” an amount sufficient to achieve astatistically significant promotion of tissue or cell regeneration, suchas muscle cell regeneration, compared to a control. In particular,indicators of muscular health, such as muscle cell regeneration,maintenance, or repair, can be assessed through various means, includingmonitoring markers of muscle regeneration, such as transcription factorssuch as Pax7, Pax3, MyoD, MRF4, and myogenin. For example, increasedexpression of such markers can indicate that muscle regeneration isoccurring or has recently occurred. Markers of muscle regeneration, suchas expression of embryonic myosin heavy chain (eMyHC), can also be usedto gauge the extent of muscle regeneration, maintenance, or repair. Forexample, the presence of eMyHC can indicate that muscle regeneration hasrecently occurred in a subject.

Muscle cell regeneration, maintenance, or repair can also be monitoredby determining the girth, or mean cross sectional area, of muscle cellsor density of muscle fibers. Additional indicators of muscle conditioninclude muscle weight and muscle protein content. Mitotic index (such asby measuring BrdU incorporation) and myogenesis can also be used toevaluate the extent of muscle regeneration.

In particular examples, the improvement in muscle condition, such asregeneration, compared with a control is at least about 10%, such as atleast about 30%, or at least about 50% or more.

Tissue: An aggregate of cells, usually of a particular kind, togetherwith their intercellular substance that form one of the structuralmaterials of an animal and that in animals include connective tissue,epithelium, muscle tissue, and nerve tissue.

Treating a disease: A therapeutic intervention that ameliorates a signor symptom of a disease or pathological condition related to a musculardystrophy, such as a sign or symptom of muscular dystrophy. Treatmentcan induce remission or cure of a condition or slow progression, forexample, in some instances can include inhibiting the full developmentof a disease, for example preventing development of a musculardystrophy. Prevention of a disease does not require a total absence ofdisease. For example, a decrease of at least 20%, such as at least 30%,at least 40%, at least 50%, decrease in a sign or symptom associatedwith the condition or disease, such as MD, can be sufficient. As usedherein, the term “ameliorating,” with reference to a disease orcondition, refers to any observable beneficial effect of the treatment.The beneficial effect can be evidenced, for example, by a delayed onsetof clinical symptoms of the disease or condition in a susceptiblesubject, a reduction in severity of some or all clinical symptoms of thedisease or condition, a slower progression of the disease or condition,a reduction in the number of relapses of the disease or condition, animprovement in the overall health or well-being of the subject, by otherparameters well known in the art that are specific to the particulardisease or condition, and combinations of such factors.

III. Methods of Diagnosing and Prognosing Muscular Dystrophy

Methods are disclosed for diagnosing and prognosing muscular dystrophy,such as DMD, LGMD, FHMD, Beckers muscular dystrophy (BMD) or MDC1A, in asubject. In one example, the methods include detecting expression of atleast one (such as at least 2, at least 3, at least 4, at least 5, atleast 6, at least 7, at least 8, at least 30, at least 50, at least 80,at least 100, at least 190 or more) muscular dystrophy-associatedmolecules in a sample obtained from a subject either at risk of havingor having one or more signs or symptoms associated with musculardystrophy. In some examples, the muscular dystrophy-associated moleculescan include, consist essentially of, or consist of disintegrin andmetalloproteinase with thrombospondin motifs 5 (Adamts5), agrin (Agrn),collagen 6A1 (Col6a1), Galectin-1, Galectin-3, matrix metalloproteinase2 (Mmp2), integrin α3 (Iga3), integrin α6 (Iga6), integrin α7 (Iga7),laminin-α4 (Lama4), laminin-α5 (Lama5), nidogen 1 (Nid1), tenascin C(Tnc), tissue inhibitor of metalloproteinase 1 (Timp1), tissue inhibitorof metalloproteinase 2 (Timp2) or any combination thereof. In someexamples, muscular dystrophy-associated molecules include Galectin-1,Galectin-3, Col6A1, Itga3, Iga6, Itga7, Tnc and Timp 1. In someexamples, muscular dystrophy-associated molecules include Galectin-1 andGalectin-3. In some examples, muscular dystrophy-associated moleculesinclude Galectin-3 and Tnc. In some examples, the musculardystrophy-associated molecules include at least Galectin-3 for detectingDMD, LGMD, FHMD, BMD or MDC1A. In some examples, the musculardystrophy-associated molecules include at least Galectin-3 for detectingDMD. “Consists essentially of” in this context indicates that theexpression of additional molecules can be evaluated (such as a control),but that these molecules do not include more than the listed musculardystrophy-associated molecules. Thus, in one example, the expression ofa control, such as a housekeeping protein or rRNA can be assessed (suchas 18S RNA, beta-microglobulin, GAPDH, and/or 18S rRNA). In someexamples, “consist essentially of” indicates that no more than 5 othermolecules are evaluated, such as no more than 4, 3, 2, or 1 othermolecules. In this context “consist of” indicates that only theexpression of the stated molecules are evaluated; the expression ofadditional molecules is not evaluated.

The methods also can include comparing expression of the at least onemuscular dystrophy-associated molecule in the sample obtained from thesubject at risk of having or having one or more signs or symptomsassociated with muscular dystrophy to a control, wherein an increase inthe expression of the at least one muscular dystrophy-associatedmolecule relative to the control indicates that the subject has adecreased chance of survival. For example, an increase in the expressionof Galectin-3 relative to a normal control sample or reference value (orrange of values) indicates a poor prognosis, such as a decreased chanceof survival. In an example, a decreased chance of survival includes asurvival time of equal to or less than 50 months, such as 40 months, 30months, 20 months, 12 months, 6 months or 3 months from time ofdiagnosis. Conversely, a decrease in expression of a musculardystrophy-associated molecule or expression levels similar to those incontrol levels indicates a better prognosis, such as an increased chanceof survival (e.g., survival time of at least 50 months from time ofdiagnosis, such as 60 months, 80 months, 100 months, 120 months or 150months from time of diagnosis). For example, the level of the musculardystrophy-associated molecule detected can be compared to a control orreference value, such as a value that represents a level of a musculardystrophy-associated molecule expected if a subject does not havemuscular dystrophy. In one example, the muscular dystrophy-associatedmolecule detected in the sample obtained from the subject beingevaluated is compared to the level of such molecules detected in asample obtained from a subject that does not have muscular dystrophy. Incertain examples, detection of at least a 2-fold, such as at least3-fold, at least 4-fold, at least 6-fold or at least 10-fold increase inthe relative amount of the muscular dystrophy-associated molecule in thetest sample, as compared to the relative amount of such molecules in acontrol, indicates that the subject has muscular dystrophy, such as DMD,LGMD, or MDC1A, has a poor prognosis (e.g., survival time of less than50 months from time of diagnosis, such as 40 months, 30 months, 20months, 12 months, 6 months or 3 months from time of diagnosis orincreased muscle deterioration), or combinations thereof. In someexamples, detection of statistically similar relative amounts (ordecreased amounts) of muscular dystrophy-associated molecules observedin a test sample, as compared to the relative amount of such moleculesin a control sample, indicates that that subject does not have musculardystrophy, such as DMD, LGMD, FHMD, BMD or MDC1A, has a good prognosis(survival time of at least 50 months from time of diagnosis, such as 60months, 80 months, 100 months, 120 months or 150 months from time ofdiagnosis), or combinations thereof.

Alterations in the expression can be measured at the nucleic acid level(such as by real time quantitative polymerase chain reaction ormicroarray analysis) or at the protein level (such as by Western blotanalysis or ELISA).

In some examples, such methods can be used to identify those subjectsthat will benefit from the disclosed treatment methods. For example,such diagnostic or prognostic methods can be performed prior to thesubject undergoing the treatment. In other examples, these methods areutilized to predict subject survival or the efficacy of a giventreatment, or combinations thereof. Thus, the methods of the presentdisclosure are valuable tools for practicing physicians to make quicktreatment decisions regarding how to treat muscular dystrophy, such asDMD, LGMD, FHMD, BMD or MDC1A. These treatment decisions can include theadministration of an agent for treating one or more signs or symptomsassociated with muscular dystrophy and decisions to monitor a subjectfor onset and/or advancement of a muscular dystrophy associatedcondition. The method disclosed herein can also be used to monitor theeffectiveness of a therapy.

Following the measurement of the expression levels of one or more of themolecules identified herein, the assay results, findings, diagnoses,predictions and/or treatment recommendations are typically recorded andcommunicated to technicians, physicians and/or patients, for example. Incertain embodiments, computers will be used to communicate suchinformation to interested parties, such as, patients and/or theattending physicians. Based on the measurement, the therapy administeredto a subject can be modified.

In one embodiment, a diagnosis, prediction and/or treatmentrecommendation based on the expression level in a test subject of one ormore of the muscular dystrophy-associated molecules disclosed herein iscommunicated to the subject as soon as possible after the assay iscompleted and the diagnosis and/or prediction is generated. The resultsand/or related information may be communicated to the subject by thesubject's treating physician. Alternatively, the results may becommunicated directly to a test subject by any means of communication,including writing, such as by providing a written report, electronicforms of communication, such as email, or telephone. Communication maybe facilitated by use of a computer, such as in case of emailcommunications. In certain embodiments, the communication containingresults of a diagnostic test and/or conclusions drawn from and/ortreatment recommendations based on the test, may be generated anddelivered automatically to the subject using a combination of computerhardware and software which will be familiar to artisans skilled intelecommunications. One example of a healthcare-oriented communicationssystem is described in U.S. Pat. No. 6,283,761; however, the presentdisclosure is not limited to methods which utilize this particularcommunications system. In certain embodiments of the methods of thedisclosure, all or some of the method steps, including the assaying ofsamples, diagnosing of diseases, and communicating of assay results ordiagnoses, may be carried out in diverse (e.g., foreign) jurisdictions.

In several embodiments, identification of a subject as having musculardystrophy, such as DMD, LGMD, FHMD, BMD or MDC1A, results in thephysician treating the subject, such as prescribing one or more agentsfor inhibiting or delaying one or more signs and symptoms associatedwith muscular dystrophy. In additional embodiments, the dose or dosingregimen is modified based on the information obtained using the methodsdisclosed herein.

Detection of Muscular Dystrophy-Associated Nucleic Acids

In one example, one or more muscular dystrophy-associated molecules canbe detected by polymerase chain reaction (PCR). The biological samplecan be incubated with primers that permit the amplification of one ormore of the disclosed muscular dystrophy, such as DMD, LGMD, orMDC1A-associated mRNAs, under conditions sufficient to permitamplification of such products.

In another example, the biological sample is incubated with probes thatcan bind to one or more of the disclosed muscular dystrophy-associatednucleic acid sequences (such as cDNA, genomic DNA, or RNA (such asmRNA)) under high stringency conditions. The resulting hybridization canthen be detected using methods known in the art, such as by Northernblot analysis.

In an example, the isolated nucleic acid molecules or amplificationproducts are incubated with an array including oligonucleotidescomplementary to at least one muscular dystrophy-associated molecule,such as disintegrin and metalloproteinase with thrombospondin motifs 5(Adamts5), agrin (Agrn), collagen 6A1 (Col6a1), Galectin-1, Galectin-3,matrix metalloproteinase 2 (Mmp2), integrin α3 (Iga3), integrin α6(Iga6), integrin α7 (Iga7), laminin-α4 (Lama4), laminin-α5 (Lamas),nidogen 1 (Nid1), tenascin C (Tnc), tissue inhibitor ofmetalloproteinase 1 (Timp1), tissue inhibitor of metalloproteinase 2(Timp2) or any combination thereof for a time sufficient to allowhybridization between the isolated nucleic acid molecules andoligonucleotide probes, thereby forming isolated nucleic acid molecule:oligonucleotide complexes. The isolated nucleic acidmolecule:oligonucleotide complexes are then analyzed to determine ifexpression of the isolated nucleic acid molecules is altered.

In some examples, oligonucleotides complementary to Galectin-1,Galectin-3, Col6A1, Itga3, Iga6, Itga7, Tnc and Timp 1 are includedwithin the array. In some examples, an array includes oligonucleotidescomplementary to at least Galectin-1 and Galectin-3. In some examples,an array includes oligonucleotides complementary to at least Galectin-3and Tnc. In some examples, an array includes oligonucleotidescomplementary to at least Galectin-3 for detecting DMD, LGMD, FHMD, BMDor MDC1A. In some examples, an array includes oligonucleotidescomplementary to at least Galectin-1 for detecting DMD. . In someexamples, an array includes oligonucleotides complementary to at leastGalectin-1 for detecting DMD, LGMD, FHMD, BMD or MDC1A. In someexamples, an array includes oligonucleotides complementary to at leastGalectin-1 for detecting DMD.

Detecting Muscular Dystrophy-Associated Proteins

As an alternative to analyzing the sample for the presence of nucleicacids, alterations in protein expression can be measured by methodsknown in the art, such as by Western blot analysis, immunoassay (e.g.,ELISA), mass spectrometry or a protein microarray. For example, thepresence of one or more muscular dystrophy-associated molecules can bedetermined by using a protein array that includes one or more captureagents, such as antibodies that are specific for the one or moredisclosed muscular dystrophy-associated molecules.

In one example, the antibody that specifically binds a musculardystrophy-associated molecule (such as Galectin-1 or Galectin-3) isdirectly labeled with a detectable label. In another example, eachantibody that specifically binds a muscular dystrophy-associatedmolecule (the first antibody) is unlabeled and a second antibody orother molecule that can bind the human antibody that specifically bindsthe respective muscular dystrophy-associated molecule is labeled. As iswell known to one of skill in the art, a second antibody is chosen thatis able to specifically bind the specific species and class of the firstantibody. For example, if the first antibody is a human IgG, then thesecondary antibody can be an anti-human-IgG. Other molecules that canbind to antibodies include, without limitation, Protein A and Protein G,both of which are available commercially.

Suitable labels for the antibody or secondary antibody include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, magnetic agents and radioactive materials. Non-limitingexamples of suitable enzymes include horseradish peroxidase, alkalinephosphatase, beta-galactosidase, or acetylcholinesterase. Non-limitingexamples of suitable prosthetic group complexes includestreptavidin/biotin and avidin/biotin. Non-limiting examples of suitablefluorescent materials include umbelliferone, Cy3, Cy5, fluorescein,fluorescein isothiocyanate, rhodamine, dichlorotriazinylaminefluorescein, dansyl chloride or phycoerythrin. A non-limiting exemplaryluminescent material is luminol; a non-limiting exemplary magnetic agentis gadolinium, and non-limiting exemplary radioactive labels include¹²⁵I, ¹³¹I, ³⁵S or ³H.

In some examples, the presence of one or more musculardystrophy-associated molecules can be determined by using an ELISA.ELISA is a heterogeneous immunoassay, which has been widely used inlaboratory practice since the early 1970s, and can be used in themethods disclosed herein. The assay can be used to detect proteinantigens in various formats. In the “sandwich” format the antigen beingassayed is held between two different antibodies. In this method, asolid surface is first coated with a solid phase antibody. The testsample, containing the antigen (e.g., a diagnostic protein), or acomposition containing the antigen, such as a urine sample from asubject of interest, is then added and the antigen is allowed to reactwith the bound antibody. Any unbound antigen is washed away. A knownamount of enzyme-labeled antibody is then allowed to react with thebound antigen. Any excess unbound enzyme-linked antibody is washed awayafter the reaction. The substrate for the enzyme used in the assay isthen added and the reaction between the substrate and the enzymeproduces a color change. The amount of visual color change is a directmeasurement of specific enzyme-conjugated bound antibody, andconsequently the antigen present in the sample tested.

In an alternative example, muscular dystrophy-associated molecules canbe assayed in a biological sample by a competition immunoassay utilizingmuscular dystrophy-associated molecule standards labeled with adetectable substance and unlabeled antibody that specifically bind tothe desired muscular dystrophy-associated molecule. In this assay, thebiological sample (such as serum, tissue biopsy, or cells isolated froma tissue biopsy), the labeled muscular dystrophy-associated moleculestandards and the antibody that specifically binds to the musculardystrophy-associated molecule are combined and the amount of labeledmuscular dystrophy-associated molecule standard bound to the unlabeledantibody is determined. The amount of muscular dystrophy-associatedmolecule in the biological sample is inversely proportional to theamount of labeled muscular dystrophy-associated molecule standard boundto the antibody that specifically binds the musculardystrophy-associated molecule.

In some examples, ELISA can also be used as a competitive assay. In thecompetitive assay format, the test specimen containing the antigen to bedetermined is mixed with a precise amount of enzyme-labeled antigen andboth compete for binding to an anti-antigen antibody attached to a solidsurface. Excess free enzyme-labeled antigen is washed off before thesubstrate for the enzyme is added. The amount of color intensityresulting from the enzyme-substrate interaction is a measure of theamount of antigen in the sample tested. A heterogenous immunoassay, suchas an ELISA, can be used to detect any molecules associated withmuscular dystrophy.

The methods as disclosed herein, such as with a method diagnosing asubject with MD or determining the effectiveness of a particulartreatment, can be performed manually or automatically, for example on anautomated sample processing instrument with capability of detectingnucleic acid and protein sequences and comparing expression levels ofsuch sequences. Automated systems typically are at least partially, ifnot substantially entirely, under computer control. Because automatedsystems typically are at least partially computer controlled, certainembodiments of the present disclosure also concern one or more tangiblecomputer-readable media that stores computer-executable instructions forcausing a computer to perform disclosed embodiments of the method. Thus,disclosed are computers or tangible computer readable medium withinstructions for the disclose methods. Tangible computer readable mediummeans any physical object or computer element that can store and/orexecute computer instructions. Examples of tangible computer readablemedium include, but not limited to, a compact disc (CD), digitalversatile disc (DVD), blu-ray disc (BD), usb floppy drive, floppy disk,random access memory (RAM), read-only memory (ROM), erasableprogrammable read-only memory (EPROM), optical fiber, and the like. Itshould be noted that the tangible computer readable medium may even bepaper or other suitable medium in which the instructions can beelectronically captured, such as optical scanning. Where opticalscanning occurs, the instructions may be compiled, interpreted, orotherwise processed in a suitable manner, if necessary, and then storedin computer memory.

Alternatively, it may be a plugin or part of a software code that can beincluded in, or downloaded and installed into a computer application. Asa plugin, it may be embeddable in any kind of computer document, such asa webpage, word document, pdf file, mp3 file, etc.

An exemplary computer system for implementing a disclosed method, suchas with a method diagnosing a subject with MD or determining theeffectiveness of a particular treatment, includes a computer (such as apersonal computer, laptop, palmtop, set-top, server, mainframe, handheld device, and other varieties of computer), including a processingunit, a system memory, and a system bus that couples various systemcomponents including the system memory to the processing unit. Theprocessing unit can be any of various commercially available processors,including INTEL® ×86, PENTIUM® and compatible microprocessors fromINTEL® and others, including Cyrix, AMD and Nexgen; Alpha from Digital;MIPS from MIPS Technology, NEC, IDT®, Siemens, and others; and thePowerPC from IBM® and Motorola. Dual microprocessors and othermulti-processor architectures also can be used as the processing unit121.

The system bus can be any of several types of bus structure including amemory bus or memory controller, a peripheral bus, and a local bus usingany of a variety of conventional bus architectures such as PCI, VESA,AGP, Microchannel, ISA and EISA, to name a few. A basic input/outputsystem (BIOS), containing the basic routines that help to transferinformation between elements within the computer, such as duringstart-up, is stored in ROM. The system memory includes read only memoryand random access memory (RAM).

The computer may further include a hard disk drive, a magnetic diskdrive, for example to read from or write to a removable disk, and anoptical disk drive, for example to read a CD-ROM disk or to read from orwrite to other optical media. The hard disk drive, magnetic disk drive,and optical disk drive are connected to the system bus by a hard diskdrive interface, a magnetic disk drive interface, and an optical driveinterface, respectively. The drives and their associated computerreadable media provide nonvolatile storage of data, data structures(databases), computer executable instructions, etc. for the computer.Although the description of computer readable media above refers to ahard disk, a removable magnetic disk and a CD, it should be appreciatedby those skilled in the art that other types of media which are readableby a computer, such as magnetic cassettes, flash memory cards, digitalvideo disks, Bernoulli cartridges, and the like, can also be used in theexemplary operating environment.

A user can enter commands and information into the computer usingvarious input devices, such as a keyboard and pointing device, such as amouse. Other input devices can include a microphone, satellite dish,scanner, or the like. These and other input devices are often connectedto the processing unit through a serial port interface that is coupledto the system bus, but can be connected by other interfaces, such as aparallel port, game port or a universal serial bus (USB). A monitor orother type of display device is also connected to the system bus via aninterface, such as a video adapter. In addition to the monitor,computers typically include other peripheral output devices, such asprinters.

The computer can operate in a networked environment using logicalconnections to one or more other computer systems, such as computer. Theother computer systems can be servers, routers, peer devices or othercommon network nodes, and typically include many or all of the elementsdescribed relative to the computer. Logical connections can include alocal area network (LAN) and a wide area network (WAN). Such networkingenvironments are common in offices, enterprise-wide computer networks,intranets and the Internet.

When used in a LAN networking environment, the computer is connected tothe local network through a network interface or adapter. When used in aWAN networking environment, the computer 120 typically includes a modemor other means for establishing communications (for example via the LANand a gateway or proxy server) over the wide area network, such as theInternet. The modem, which can be internal or external, is connected tothe system bus via the serial port interface. In a networkedenvironment, program modules depicted relative to the computer, orportions thereof, can be stored in the remote memory storage device. Itwill be appreciated that the network connections shown are exemplary andother means of establishing a communications link between the computersystems (including an Ethernet card, ISDN terminal adapter, ADSL modem,10 BaseT adapter, 100 BaseT adapter, ATM adapter, or the like) can beused.

The methods, including the acts and operations they comprise, describedabove can be performed by the computer. Such acts and operations aresometimes referred to as being computer executed. It will be appreciatedthat the acts and symbolically represented operations include themanipulation by the processing unit of electrical signals representingdata bits which causes a resulting transformation or reduction of theelectrical signal representation, and the maintenance of data bits atmemory locations in the memory system (including the system memory, harddrive, floppy disks, and CD-ROM) to thereby reconfigure or otherwisealter the computer system's operation, as well as other processing ofsignals. The memory locations where data bits are maintained arephysical locations that have particular electrical, magnetic, or opticalproperties corresponding to the data bits.

It is contemplated that a distributed computing environment can be usedto implement the methods and systems of the present disclosure mayreside. The distributed computing environment includes two computersystems connected by a connection medium, although the disclosed methodis equally applicable to an arbitrary, larger number of computer systemsconnected by the connection medium. The computer systems can be any ofseveral types of computer system configurations, including personalcomputers, multiprocessor systems, handheld devices, and the like. Interms of logical relation with other computer systems, a computer systemcan be a client, a server, a router, a peer device, or other commonnetwork node. Additional computer systems may be connected by anarbitrary number of connection mediums. The connection medium cancomprise any local area network (LAN), wide area network (WAN), or othercomputer network, including but not limited to Ethernets,enterprise-wide computer networks, intranets and the Internet.

Portions of the software for automated gene detection and quantificationas well as databases storing correlation data can be implemented in asingle computer system, with the application later distributed to othercomputer systems in the distributed computing environment. Portions ofthe software for determining gene expression and quantification may alsobe practiced in a distributed computing environment where tasks areperformed by a single computer system acting as a remote processingdevice that is accessed through a communications network, with thedistributed application later distributed to other computer systems inthe distributed computing environment. In a networked environment,program modules comprising the software for determining gene expressionand quantification as well as databases storing the correlation data canbe located on more than one computer system. Communication between thecomputer systems in the distributed computing network may advantageouslyinclude encryption of the communicated data.

In certain embodiments, the communication containing results of adiagnostic test and/or conclusions drawn from and/or treatmentrecommendations based on the test, may be generated and deliveredautomatically to the subject, facility, physician and the like using acombination of computer hardware and software which will be familiar toartisans skilled in telecommunications. One example of ahealthcare-oriented communications system is described in U.S. Pat. No.6,283,761; however, the present disclosure is not limited to methodswhich utilize this particular communications system. In certainembodiments of the methods of the disclosure, all or some of the methodsteps, including the assaying of samples, diagnosing of diseases, andcommunicating of assay results or diagnoses, may be carried out indiverse (e.g., foreign) jurisdictions.

IV. Methods of Use

It is shown herein that muscular dystrophy is associated withdifferential expression of muscular dystrophy-associated molecules, suchas disintegrin and metalloproteinase with thrombospondin motifs 5(Adamts5), agrin (Agrn), collagen 6A1 (Col6a1), Galectin-1, Galectin-3,matrix metalloproteinase 2 (Mmp2), integrin α3 (Iga3), integrin α6(Iga6), integrin α7 (Iga7), laminin-α4 (Lama4), laminin-α5 (Lama5),nidogen 1 (Nid1), tenascin C (Tnc), tissue inhibitor ofmetalloproteinase 1 (Timp1), tissue inhibitor of metalloproteinase 2(Timp2). Based on these observations, methods of treatment to reduce oreliminate one or more signs or symptoms associated with musculardystrophy, such as DMD, LGMD, FHMD, BMD or MDC1A are disclosed bydecreasing the expression of at least one of the disclosed musculardystrophy-associated molecules. In a particular example, the subject isa human.

Methods are disclosed herein for treating muscular dystrophy, such asDMD, LGMD, FHMD, BMD or MDC 1 A. In one example, the method includesadministering an effective amount of an agent to a subject with musculardystrophy in which the agent alters the biological activity orexpression of one or more of the disclosed muscular dystrophy-associatedmolecules, such as one or more of disintegrin and metalloproteinase withthrombospondin motifs 5 (Adamts5), agrin (Agrn), collagen 6A1 (Col6a1),Galectin-1, Galectin-3, matrix metalloproteinase 2 (Mmp2), integrin α3(Iga3), integrin α6 (Iga6), integrin α7 (Iga7), laminin-α4 (Lama4),laminin-α5 (Lama5), nidogen 1 (Nid1), tenascin C (Tnc), tissue inhibitorof metalloproteinase 1 (Timp1), tissue inhibitor of metalloproteinase 2(Timp2). Such agents can alter the expression of nucleic acid sequences(such as DNA, cDNA, or mRNAs) and proteins. In some examples, an agentdecreases alters the biological activity or expression of one or more ofthe disclosed muscular dystrophy-associated molecules. A decrease in theexpression does not need to be 100% for the composition to be effective.For example, an agent can decrease the expression or biological activityby a desired amount, for example by at least 20%, at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 95%, at least98%, or even at least 100% as compared to activity or expression in acontrol. In some examples, an agent increases the biological activity orexpression of one or more of the disclosed muscular dystrophy-associatedmolecules

In particular examples, the agent is a specific binding agent that bindsto and decreases the expression of one or more of the disclosed musculardystrophy-associated molecules. Specific molecules include disintegrinand metalloproteinase with thrombospondin motifs 5 (Adamts5), agrin(Agrn), collagen 6A1 (Col6a1), galectin, such as Galectin-3, matrixmetalloproteinase 2 (Mmp2), integrin α3 (Iga3), integrin α6 (Iga6),integrin α7 (Iga7), laminin-α4 (Lama4), laminin-α5 (Lamas), nidogen 1(Nid1), tenascin C (Tnc), tissue inhibitor of metalloproteinase 1(Timp1), tissue inhibitor of metalloproteinase 2 (Timp2) as well asfragments of the full-length molecules, cDNAs, or mRNAs (and proteinsencoded thereby) whose expression is increased in response to musculardystrophy, such as DMD, LGMD, FHMD, BMD or MDC1A. The agents can alterthe activity or expression of the one or more disclosed musculardystrophy-associated molecules well as other molecules involved inmuscular dystrophy progression.

In particular examples, the agent is an inhibitor such as a siRNA or anantibody to one of the disclosed muscular dystrophy-associated moleculesthat is upregulated in muscular dystrophy patients. For example, theagent can be an siRNA that interferes with mRNA expression of one of thedisclosed muscular dystrophy-associated molecules. For example, theagent is an siRNA that inhibitor reduces expression of one or more ofthe disclosed muscular dystrophy-associated molecules. In additionalexamples, a composition includes at least two agents such as twospecific siRNAs that each bind to their respective musculardystrophy-associated nucleotide sequences and inhibit one or more signsor symptoms associated with muscular dystrophy in the subject. In someexamples, the agent is an activator or agonist that is used to stimulateor increase the biological activity or expression of one of thedisclosed muscular dystrophy-associated molecules.

Also disclosed are methods of increasing/maintaining muscle strengthand/or bone density. In some examples, an effective amount of galectinor galectin composition, such as galectin-1 composition, is administeredto increase muscle strength and/or bone density and/or prevent, inhibitor slow muscle and bone loss. In some examples, galectin or a galectincomposition, such as galectin-1 composition, is administered to asubject at risk of muscle/bone injury or muscle/bone loss, such as to anathlete, an astronaut, or any other individual that partakes inactivities that may cause muscle/bone injury and/or loss. In someexamples, a disclosed regimen, such as a disclosed galectin-1 regimen,is provided to prevent muscle/bone injury and/or loss. In some examples,a disclosed regimen, such as a disclosed galectin-1 regimen, is providedto maintain bone density and/or muscle strength. In some examples, adisclosed regimen, such as a disclosed galectin-1 regimen, is providedto treat a subject experiencing a loss in bone density and/or musclestrength whereby the regimen is administered in a manner to lead to anincrease in bone density and/or muscle strength or to maintain theexisting muscle strength and bone density (e.g., prevent further loss ofmuscle strength and/or bone density).

In some examples, galectin or a galectin composition, such as aGalectin-1 composition, is administered to a subject at risk ofacquiring or suffering from a condition or disease associated withmuscle loss, bone loss, muscle density loss, and/or muscle strengthloss, such as, but not limited to a subject at risk of acquiring orsuffering from kyphosis, muscular dystrophies, scoliosis, broken bones,muscle strains, muscle tears, tendon injury, osteoporosis, rheumatoidarthritis, lupus, scoliosis and/or multiple sclerosis. In some examples,galectin or a galectin composition, such as a Galectin-1 composition, isadministered for preventing, treating or slowing the progression of asign or symptom associated with aging. It is contemplated that galectinor a galectin composition can be administered for short or prolongperiods of time, ranging from days to years. In some examples, galectinor a galectin composition, such as Galectin-1 or a Galectin-1composition, is administered post-surgery, such as to a subject that hasundergone surgery and may be at risk of experiencing or has muscle loss,bone loss, muscle strength loss or muscle density loss. In someexamples, a galectin composition, such as Galectin-1, is administered toa female subject post-pregnancy. In some examples, a galectincomposition, such as Galectin-1, is administered to a short or long-termcoma subject.

Agents

Desirable agents are those that when administered in effective amountsinduce the desired response (e.g., prevent, inhibit, or treat one ormore signs of a particular condition or disease, including but notlimited to kyphosis, muscular dystrophies, scoliosis, broken bones,muscle strains, muscle tears, tendon injury, osteoporosis, rheumatoidarthritis, lupus, scoliosis and/or multiple sclerosis). In one example,agents are specific binding agents that bind with higher affinity to amolecule of interest, than to other molecules. For example, a specificbinding agent can be one that binds with high affinity to one of thegenes or gene products of a disclosed muscular dystrophy-associatedmolecules, but does not substantially bind to another gene or geneproduct. In some examples, a specific binding agent binds to onethrombospondin motifs 5 (Adamts5), agrin (Agrn), collagen 6A1 (Col6a1),Galectin-1, Galectin-3, matrix metalloproteinase 2 (Mmp2), integrin α3(Iga3), integrin α6 (Iga6), integrin α7 (Iga7), laminin-α4 (Lama4),laminin-α5 (Lama5), nidogen 1 (Nid1), tenascin C (Tnc), tissue inhibitorof metalloproteinase 1 (Timp1), tissue inhibitor of metalloproteinase 2(Timp2) that are upregulated in muscular dystrophy subjects, therebyreducing or inhibiting expression of the gene, but does not bind to theother genes (or gene product).

In some examples, the agent interferes with gene expression(transcription, processing, translation, post-translationalmodification), such as, by interfering with the gene's mRNA and blockingtranslation of the gene product or by post-translational modification ofa gene product, or by causing changes in intracellular localization. Inanother example, a specific binding agent binds to a protein encoded byof one of the genes disclosed herein to be associated with the specificcondition or disease with a binding affinity in the range of 0.1 to 20nM and reduces or inhibits the activity of such protein. In someexamples, the agent increases gene expression (transcription,processing, translation, post-translational modification) or theactivity of the protein associated with the specific condition ordisease.

Examples of specific binding agents include siRNAs, antibodies, ligands,recombinant proteins, peptide mimetics, and soluble receptor fragments.One example of a specific binding agent is a siRNA. Methods of makingsiRNAs that can be used clinically are known in the art. ParticularsiRNAs and methods that can be used to produce and administer them aredescribed in detail below. In a specific example, a specific bindingagent includes a Galectin-3 siRNA molecule.

Another specific example of a specific binding agent is an antibody,such as a monoclonal or polyclonal antibody. Methods of makingantibodies that can be used clinically are known in the art. Particularantibodies and methods that can be used to produce them are known tothose of ordinary skill in the art. Further, antibodies to Galectin-1and Galectin-3 are commercially available.

In a further example, small molecular weight inhibitors/antagonists oractivators/agonists of the receptor protein can be used to regulateactivity such as the expression or production of musculardystrophy-associated molecules. In a particular example, small molecularweight inhibitors/antagonists or activators/agonists of the proteinsencoded by the genes of thrombospondin motifs 5 (Adamts5), agrin (Agrn),collagen 6A1 (Col6a1), Galectin-1, Galectin-3, matrix metalloproteinase2 (Mmp2), integrin α3 (Iga3), integrin α6 (Iga6), integrin α7 (Iga7),laminin-α4 (Lama4), laminin-α5 (Lama5), nidogen 1 (Nid1), tenascin C(Tnc), tissue inhibitor of metalloproteinase 1 (Timp1), and/or tissueinhibitor of metalloproteinase 2 (Timp2) are used.

Specific binding agents can be therapeutic, for example by altering thebiological activity of a nucleic acid or protein that is associatedmuscular dystrophy progression. For example, a specific binding agentthat binds with high affinity to one or more genes disclosed herein tobe upregulated in subjects with muscular dystrophy, may substantiallyreduce the biological function of the gene or gene product. In otherexamples, a specific binding agent that binds with high affinity to oneof the proteins disclosed herein to be upregulated in subjects withmuscular dystrophy, may substantially reduce the biological function ofthe protein. Such agents can be administered in effective amounts tosubjects in need thereof, such as a subject having muscular dystrophy,such as DMD, LGMD, FHMD, BMD or MDC1A.

In other examples, a specific binding agent that binds with highaffinity to one or more genes disclosed herein to be upregulated insubjects with muscular dystrophy, may substantially increase thebiological function of the gene or gene product. In other examples, aspecific binding agent that binds with high affinity to one of theproteins disclosed herein to be upregulated in subjects with musculardystrophy, may substantially increase the biological function of theprotein. Such agents can be administered in effective amounts tosubjects in need thereof, such as a subject having muscular dystrophy,such as DMD, LGMD, FHMD, BMD or MDC1A.

For example, galectin or a composition comprising galectin can betherapeutic. The present disclosure relates to a method of providingbenefit to a subject by administering to the subject a galectin or acomposition that includes galectin, such as Galectin-1 or Galectin-3. Ina particular embodiment, the present disclosure provides a method ofenhancing muscle regeneration, such as to treat muscular dystrophy, in asubject by administering galectin or a galectin composition.

In various embodiments, the present disclosure provides a method oftreating a subject with galectin or a composition that includesgalectin. For example, some embodiments provide methods of improvingmuscular health, such as enhancing muscle regeneration, maintenance, orrepair in a subject by administering to the subject an effective amountof galectin or a composition comprising galectin, including fragments,derivatives, or analogs thereof. In a specific example, the galectin isa complete galectin protein. In further examples, the galectin isselected from Galectin-1, Galectin-3, and combinations thereof. Infurther examples, the galectin or galectin composition includes asubstance at least substantially homologous to Galectin-1 or Galectin-3.In yet further implementations, the galectin or galectin compositioncomprises a polypeptide at least substantially homologous to theGalectin-1 or Galectin-3.

In additional examples, the galectin or galectin composition consists ofGalectin-1, Galectin-3, and combinations thereof. In further examples,the galectin or galectin composition consists of a substance at leastsubstantially homologous to Galectin-1 or Galectin-3. In a specificexample, the galectin or galectin composition does not include agalectin fragment, such as including only a complete galectin protein.

In yet another example, the galectin or galectin composition consistsessentially of Galectin-1, Galectin-3, and combinations thereof. Infurther examples, the galectin or galectin composition consistsessentially of a substance at least substantially homologous toGalectin-1 or Galectin-3. In yet further implementations, the galectinor galectin composition consists essentially of a polypeptide at leastsubstantially homologous to the galectin α1 chain. In a specificexample, the galectin or galectin composition does not include agalectin fragment, such as including essentially only a completegalectin protein.

Further implementations of the disclosed method include diagnosing thesubject as having a condition treatable by administering galectin or acomposition comprising galectin, such as by administering Galectin-1,Galectin-3 or a combination thereof or a composition containingGalectin-1, Galectin-3 or a combination. In one example, the subject isdiagnosed as suffering from muscular dystrophy, such as LGMD, FHMD,Beckers muscular dystrophy and/or MDC1A. In further instances thecondition is characterized by the failure of a subject, or the reducedability of the subject, to express one or more proteins associated withthe formation or maintenance of the extracellular matrix, such asimpaired or non-production of a galectin, an integrin, dystrophin,utrophin, or dystroglycan.

In a specific embodiment, the present disclosure also provides a methodfor increasing muscle regeneration in a subject. For example, geriatricsubjects, subjects suffering from muscle disorders, and subjectssuffering from muscle injury, including activity induced muscle injury,such as injury caused by exercise, may benefit from this embodiment.

In yet further embodiments of the disclosed method, the galectin orgalectin composition, such as Galectin-1, Galectin-3 or a combinationthereof containing composition, is administered in a preventativemanner, such as to prevent or reduce muscular and/or bone damage orinjury (such as activity or exercise induced injury). For example,geriatric subjects, subjects prone to muscle damage, or subjects at riskfor muscular injury, such as athletes, may be treated in order toeliminate or ameliorate muscular damage, injury, or disease.

Implementations of the present disclosure may also be used to promotewound healing. In some examples, a galectin or a composition comprisinggalectin is administered into or proximate to a wound. In furtherexamples, the substance is administered systemically. Although thesubstance is typically applied after the wound occurs, the substance canbe applied prospectively in some examples.

In further embodiments, the method of the present disclosure includesadministering the galectin or galectin composition, such as Galectin-1,Galectin-3 or a combination thereof containing composition, with one ormore additional pharmacological substances, such as a therapeutic agent.In some aspects, the additional therapeutic agent enhances thetherapeutic effect of the galectin or galectin composition. In furtheraspects, the therapeutic agent provides independent therapeutic benefitfor the condition being treated. In various examples, the additionaltherapeutic agent is a component of the extracellular matrix, such as anintegrin, dystrophin, dystroglycan, utrophin, or a growth factor. Infurther examples, the therapeutic agent reduces or enhances expressionof a substance that enhances the formation or maintenance of theextracellular matrix.

In some examples, the galectin or galectin composition is applied to aparticular area of the subject to be treated. For example, the galectinor galectin composition may be injected into a particular area to betreated, such as a muscle. In further examples, the galectin or galectincomposition is administered such that it is distributed to multipleareas of the subject, such as systemic administration or regionaladministration.

Galectin, or a composition comprising galectin, such as Galectin-1,Galectin-3, or a combination thereof, can be administered by anysuitable method, such as topically, parenterally (such as intravenouslyor intraperitoneally), or orally. In a specific example, the galectin orgalectin composition is administered systemically, such as throughparenteral administration, such as stomach injection or peritonealinjection.

Although the disclosed methods generally have been described withrespect to muscle regeneration, the disclosed methods also may be usedto enhance repair or maintenance, or prevent damage to, other tissuesand organs. For example, the methods of the present disclosure can beused to treat symptoms of muscular dystrophy stemming from effects tocells or tissue other than skeletal muscle, such as impaired or alteredbrain function, smooth muscles, or cardiac muscles.

Pre-Screening Therapeutic Agents

In some examples, potential therapeutic agents are initially screenedfor treating muscular dystrophy, such as DMD, LGMD, FHMD, BMD or MDC1A,by detecting one or more muscular dystrophy-associated molecules (asdiscussed in detail below in Section VI.). For example, the disclosedmuscular dystrophy-associated molecules can be used to identify agentscapable of reducing or inhibiting one or more signs or symptoms ofmuscular dystrophy. In an example, subjects can be first pre-screenedfor the presence of muscular dystrophy, such as DMD, LGMD, FHMD, BMD orMDC1A, which will respond to a particular therapeutic agent prior toreceiving treatment.

Administration

Methods of administration of the disclosed compositions are routine, andcan be determined by a skilled clinician. For example, the disclosedtherapies (such as those that include a binding agent specific for oneof the disclosed muscular dystrophy-associated molecules or a galectin,such as Galectin-1) can be administered via injection, orally,topically, transdermally, parenterally, or via inhalation or spray. In aparticular example, a composition is administered intravenously to amammalian subject, such as a human. In another example, the compositionis administered orally. In some examples, the composition is applied toa particular are of the subject to be treated. For example, thecomposition is injected into a muscle.

The therapeutically effective amount of the agents administered can varydepending upon the desired effects and the subject to be treated. In oneexample, the method includes daily administration of at least 1 μg of atherapeutic agent to the subject (such as a human subject). For example,a human can be administered at least 1 μg or at least 1 mg of the agentdaily, such as 10 μg to 100 μg daily, 100 μg to 1000 μg daily, forexample 10 μg daily, 100 μg daily, or 1000 μg daily. In one example, thesubject is administered at least 1 μg (such as 1-100 μg) intravenouslyof the agent (such as a composition that includes a binding agent thatspecifically binds to one of the disclosed muscular dystrophy-associatedmolecules or a galectin, such as Galectin-1 or Galectin-3). In oneexample, the subject is administered at least 1 mg intramuscularly (forexample in an extremity) of such composition. The dosage can beadministered in divided doses (such as 2, 3, or 4 divided doses perday), or in a single dosage daily.

In particular examples, the subject is administered the therapeuticcomposition that includes a binding agent specific for one of thedisclosed muscular dystrophy-associated molecules or a galectin, such asGalectin-1, Galectin-3 or a combination thereof, on a multiple dailydosing schedule, such as at least two consecutive days, 10 consecutivedays, and so forth, for example for a period of weeks, months, or years.In one example, the subject is administered the therapeutic compositionthat includes a binding agent specific for one of the disclosed musculardystrophy-associated molecules or a galectin, such as Galectin-1,Galectin-3 or a combination thereof daily for a period of at least 30days, such as at least 2 months, at least 4 months, at least 6 months,at least 12 months, at least 24 months, or at least 36 months.

The compositions, such as those that include a binding agent specificfor one of the muscular dystrophy-associated molecules or a galectin(such as Galectin-1, Galectin-3 or a combination thereof), can furtherinclude one or more biologically active or inactive compounds (or both),such as other agents known in the art for reducing or treating one ormore signs or symptoms associated with muscular dystrophy andconventional non-toxic pharmaceutically acceptable carriers,respectively. For example, additional therapeutic agent which enhancethe therapeutic effect of the disclosed compositions are included, suchas a component of the extracellular matrix, such as an integrin,dystrophin, dystroglycan, utrophin, or a growth factor. In furtherexamples, the additional therapeutic agent reduces or enhancesexpression of a substance that enhances the formation or maintenance ofthe extracellular matrix. In some examples, the additional substance caninclude aggrecan, angiostatin, cadherins, collagens (including collagenI, collagen III, or collagen IV), decorin, elastin, enactin, endostatin,fibrin, fibronectin, osteopontin, tenascin, thrombospondin, vitronectin,and combinations thereof. Biglycans, glycosaminoglycans (such asheparin), glycoproteins (such as dystroglycan), proteoglycans (such asheparan sulfate), and combinations thereof can also be administered. Aparticular laminin can be administered with other forms of laminin,laminin analogs, laminin derivatives, or a fragment of any of theforegoing.

In some examples, growth stimulants such as cytokines, polypeptides, andgrowth factors such as brain-derived neurotrophic factor (BDNF), CNF(ciliary neurotrophic factor), EGF (epidermal growth factor), FGF(fibroblast growth factor), glial growth factor (GGF), glial maturationfactor (GMF) glial-derived neurotrophic factor (GDNF), hepatocyte growthfactor (HGF), insulin, insulin-like growth factors, kerotinocyte growthfactor (KGF), nerve growth factor (NGF), neurotropin-3 and -4, PDGF(platelet-derived growth factor), vascular endothelial growth factor(VEGF), and combinations thereof may be administered with one of thedisclosed therapies.

In a particular example, a therapeutic composition that includes atherapeutically effective amount of a therapeutic agent (such as abinding agent specific for one of the disclosed musculardystrophy-associated molecules or a galectin, such as Galectin-1,Galectin-3 or a combination thereof) further includes one or morebiologically inactive compounds. Examples of such biologically inactivecompounds include, but are not limited to: carriers, thickeners,diluents, buffers, preservatives, and carriers. The pharmaceuticallyacceptable carriers useful for these formulations are conventional (seeRemington's Pharmaceutical Sciences, by E. W. Martin, Mack PublishingCo., Easton, Pa., 19th Edition (1995)). In general, the nature of thecarrier will depend on the particular mode of administration beingemployed. For instance, parenteral formulations can include injectablefluids that include pharmaceutically and physiologically acceptablefluids such as water, physiological saline, balanced salt solutions,aqueous dextrose, glycerol or the like as a vehicle. For solidcompositions (for example, powder, pill, tablet, or capsule forms),conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate, In addition to biologically-neutral carriers, pharmaceuticalcompositions to be administered can include minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

The compositions can be formulated in a palatable form foradministration as a food additive or supplement. Such palatable formsare typically odor free or are masked or coated as is known to those ofordinary skill in the art of pharmaceutical formulation. Where thedisclosed compounds are administered orally, particularly when they areadministered as a nutritional supplement, the compounds can be mixedwith a foodstuff base. Such mixtures can be in the form of an emulsionor an admixture with solid food. In some examples, the disclosedcompositions, such as a Galectin-1 composition, can be mixed withyogurt. For example, health bars, without limitation, can be prepared bycombining various excipients, such as binders, fillers, flavorings,colorants and the like, along with one or more galectins, such asGalectin-1, and mixing to a plastic mass consistency. The mass is theneither extruded or molded to form “candy bar” shapes that are then driedor allowed to solidify to form the final product.

Alternatively, the compounds can be administered orally in a liquiddosage form as a solution, emulsion or suspension. The liquid dosageform can contain, for example, suitable solvents, preservatives,emulsifying agents, suspending agents, diluents, sweeteners, meltingagents, and coloring and flavoring agents, which are known to those ofordinary skill in the art. The compounds also can be added to liquidvitamin formulations and electrolyte containing drinks. Drinks may be inthe form of energy drinks, sports drinks, fruit drinks, citrus drinks,carbonated drinks, dry drink mixes, other suitable drink mediums orcombinations thereof.

Additional Treatments

In particular examples, prior to, during, or following administration ofan effective amount of an agent that reduces or inhibits one or moresigns or symptoms associated with muscular dystrophy, the subject canreceive one or more other therapies. In one example, the subjectreceives one or more treatments prior to administration of a disclosedagent specific for one of the disclosed muscular dystrophy-associatedmolecules or a galectin, such as galectin protein therapy (e.g.,Galectin-1 /Galectin-3 protein therapy). Examples of such therapiesinclude, but are not limited to, laminin-111 protein therapy, whichworks to stabilize the sarcolemma and reduce muscle degeneration. Insome examples, a source of muscle cells can be added to aid in muscleregeneration and repair. In some aspects of the present disclosure,satellite cells are administered to a subject in combination withlaminin therapy. U.S. Patent Publication 2006/0014287, incorporated byreference herein to the extent not inconsistent with the presentdisclosure, provides methods of enriching a collection of cells inmyogenic cells and administering those cells to a subject. In furtheraspects, stem cells, such as adipose-derived stem cells, areadministered to the subject. Suitable methods of preparing andadministering adipose-derived stem cells are disclosed in U.S. PatentPublication 2007/0025972, incorporated by reference herein to the extentnot inconsistent with the present disclosure. Additional cellularmaterials, such as fibroblasts, can also be administered, in someexamples.

V. Methods of Monitoring the Efficacy of a Treatment for MuscularDystrophy

Methods are also disclosed herein to monitor the efficacy of a treatmentfor muscular dystrophy. In some examples, the method of determining theeffectiveness of an agent for the treatment of muscular dystrophy in asubject with muscular dystrophy includes detecting one or more disclosedmuscular dystrophy-associated molecules in a sample from the subjectfollowing treatment with the agent; and comparing expression of suchmolecules following treatment to a reference value or control, whereinan alteration, such as a decrease or an increase, in the expression ofthe one or more muscular dystrophy-associated molecules followingtreatment indicates that the agent is effective for the treatment ofmuscular dystrophy in the subject. In some examples, these methodsutilize a biological fluid, such as, but not limited to urine or serum,for the detection of a molecule associated with muscular dystrophy,including, but not limited to, disintegrin and metalloproteinase withthrombospondin motifs 5 (Adamts5), agrin (Agrn), collagen 6A1 (Col6a1),Galectin-1, Galectin-3, matrix metalloproteinase 2 (Mmp2), integrin α3(Iga3), integrin α6 (Iga6), integrin α7 (Iga7), laminin-α4 (Lama4),laminin-α5 (Lama5), nidogen 1 (Nid1), tenascin C (Tnc), tissue inhibitorof metalloproteinase 1 (Timp1), tissue inhibitor of metalloproteinase 2(Timp2) or any combination thereof. The methods include detecting, ordetermining the abundance (amount) or activity of one or more moleculesassociated with muscular dystrophy, including those disclosed herein.

The disclosed methods can include detecting at least one, such as two,three, four, five, six, seven, eight, nine, ten, eleven, or moremolecules associated with muscular dystrophy. In one example, the methodincludes detecting at least one, such as two, three, four, five, six,seven, eight, nine, ten, eleven, twelve or thirteen of the followingmolecules associated with muscular dystrophy: disintegrin andmetalloproteinase with thrombospondin motifs 5 (Adamts5), agrin (Agrn),collagen 6A1 (Col6a1), Galectin-1, Galectin-3, matrix metalloproteinase2 (Mmp2), integrin α3 (Iga3), integrin α6 (Iga6), integrin α7 (Iga7),laminin-α4 (Lama4), laminin-α5 (Lama5), nidogen 1 (Nid1), tenascin C(Tnc), tissue inhibitor of metalloproteinase 1 (Timp1), tissue inhibitorof metalloproteinase 2 (Timp2). In some examples, the methods includedetecting at least Galectin-3. In some examples, the methods includedetecting at least Galectin-1.

In some embodiments, the method includes detecting a decrease, such as astatistically significant decrease, such as an at least a 1.5, 2, 3, 4,or 5 fold decrease in the amount of one or more molecules associatedwith muscular dystrophy, including at least a 1.5, 2, 3, 4, or 5 folddecrease in one or more of disintegrin and metalloproteinase withthrombospondin motifs 5 (Adamts5), agrin (Agrn), collagen 6A1 (Col6a1),Galectin-1, Galectin-3, matrix metalloproteinase 2 (Mmp2), integrin α3(Iga3), integrin α6 (Iga6), integrin α7 (Iga7), laminin-α4 (Lama4),laminin-α5 (Lama5), nidogen 1 (Nid1), tenascin C (Tnc), tissue inhibitorof metalloproteinase 1 (Timp1), tissue inhibitor of metalloproteinase 2(Timp2) as compared to a reference value.

In some embodiments, the method includes detecting a decrease, such as astatistically significant decrease, such as an at least 10% increase,including an at least 15%, at least 20%, at least 25%, at least 30%, atleast 40%, at least 45%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, including a 10% to 90% decrease, 20% to 80%decrease, 30% to 70% decrease or a 40% to 60% decrease (e.g., a 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 200% or moredecrease) in the amount of one or more molecules associated withmuscular dystrophy, including an at least a 10% decrease, including anat least 15%, at least 20%, at least 25%, at least 30%, at least 40%, atleast 45%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, including a 10% to 90% decrease, 20% to 80% decrease, 30% to70% decrease or a 40% to 60% decrease (e.g., a 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 100%, 200% or more decrease) in one or more ofdisintegrin and metalloproteinase with thrombospondin motifs 5(Adamts5), agrin (Agra), collagen 6A1 (Col6a1), Galectin-1, Galectin-3,matrix metalloproteinase 2 (Mmp2), integrin α3 (Iga3), integrin α6(Iga6), integrin α7 (Iga7), laminin-α4 (Lama4), laminin-α5 (Lama5),nidogen 1 (Nid1), tenascin C (Tnc), tissue inhibitor ofmetalloproteinase 1 (Timp1), tissue inhibitor of metalloproteinase 2(Timp2) as compared to a reference value.

In some embodiments, the method includes detecting an increase, such asa statistically significant increase, such as an at least a 1.5, 2, 3,4, or 5 fold increase in the amount of one or more molecules associatedwith muscular dystrophy, including at least a 1.5, 2, 3, 4, or 5 foldincrease in one or more of disintegrin and metalloproteinase withthrombospondin motifs 5 (Adamts5), agrin (Agrn), collagen 6A1 (Col6a1),Galectin-1, Galectin-3, matrix metalloproteinase 2 (Mmp2), integrin α3(Iga3), integrin α6 (Iga6), integrin α7 (Iga7), laminin-α4 (Lama4),laminin-α5 (Lama5), nidogen 1 (Nid1), tenascin C (Tnc), tissue inhibitorof metalloproteinase 1 (Timp1), tissue inhibitor of metalloproteinase 2(Timp2) as compared to a reference value.

In some embodiments, the method includes detecting an increase, such asa statistically significant increase, such as an at least 10% increase,including an at least 15%, at least 20%, at least 25%, at least 30%, atleast 40%, at least 45%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, including a 10% to 90% increase, 20% to 80%increase, 30% to 70% increase or a 40% to 60% increase (e.g., a 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 200% or moreincrease) in the amount of one or more molecules associated withmuscular dystrophy, including an at least a 10% increase, including anat least 15%, at least 20%, at least 25%, at least 30%, at least 40%, atleast 45%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, including a 10% to 90% increase, 20% to 80% increase, 30% to70% increase or a 40% to 60% increase (e.g., a 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, 100%, 200% or more increase) in one or more ofthe disclosed molecular dystrophy markers, such as Galectin-1 ascompared to a reference value.

In some embodiments, the methods can be performed over time, to monitorthe progression or regression of one or more signs or symptoms ofmuscular dystrophy in a subject, such as one or more signs or symptomsassociated with DMD, LGMD, FHMD, BMD or MDC1A. The method can beperformed multiple times over a specified time period, such as days,weeks, months or years. In several examples, the therapy includestreatment with an agent for muscular dystrophy. If the reference sampleis a normal sample, and the test sample reading (e.g., expression oractivity level of an evaluated muscular dystrophy-associated molecule)is essentially the same as the normal sample the subject is determinedto have an effective therapy, while if the test sample has asignificantly greater value for an evaluated musculardystrophy-associated molecule relative to the normal sample, the subjectis determined to have an ineffective therapy. Changes in the profile canalso represent the progression (or regression) of the disease process.The subject can be monitored while undergoing treatment using themethods described herein in order to assess the efficacy of thetreatment protocol. Following the measurement of the expression levelsof one or more of the molecules identified herein, the assay results,findings, diagnoses, predictions and/or treatment recommendations can berecorded and communicated to technicians, physicians and/or patients,for example. In certain embodiments, computers are used to communicatesuch information to interested parties, such as, patients and/or theattending physicians. Based on the measurement, the therapy administeredto a subject is modified. For example, the dose or dosing regimen ismodified based on the information obtained using the methods disclosedherein.

In one embodiment, a diagnosis, prediction and/or treatmentrecommendation based on the expression level in a test subject of one ormore of the muscular dystrophy-associated molecules disclosed herein iscommunicated to the subject as soon as possible after the assay iscompleted and the diagnosis and/or prediction is generated. The resultsand/or related information may be communicated to the subject by thesubject's treating physician. Alternatively, the results may becommunicated directly to a test subject by any means of communication,including writing, such as by providing a written report, electronicforms of communication, such as email, or telephone. Communication maybe facilitated by use of a computer, such as in case of emailcommunications. In certain embodiments, the communication containingresults of a diagnostic test and/or conclusions drawn from and/ortreatment recommendations based on the test, may be generated anddelivered automatically to the subject using a combination of computerhardware and software which will be familiar to artisans skilled intelecommunications. One example of a healthcare-oriented communicationssystem is described in U.S. Pat. No. 6,283,761; however, the presentdisclosure is not limited to methods which utilize this particularcommunications system. In certain embodiments of the methods of thedisclosure, all or some of the method steps, including the assaying ofsamples, diagnosing of diseases, and communicating of assay results ordiagnoses, may be carried out in diverse (e.g., foreign) jurisdictions.

VI. Methods of Identifying Agents for Treating Muscular Dystrophy

Methods are provided herein for identifying agents to treating musculardystrophy, such as DMD, LGMD, FHMD, BMD and MDC1A. In some examples, themethod of includes contacting a sample, such as a blood or urine sample,with one or more test agents under conditions sufficient for the one ormore test agents to decrease the expression or biological activity ofone or more of the disclosed muscular dystrophy-associated molecules.The method can also include detecting expression or biological activityof the one or more disclosed muscular dystrophy-associated molecules inthe presence of the one or more test agents. The expression orbiological activity of the one or more disclosed musculardystrophy-associated molecules in the presence of the one or more testagents is then compared to a control, such as a reference value todetermine if there is an alteration in expression or activity of the oneor more disclosed muscular dystrophy-associated molecules, whereindecreased activity or expression of the one or more disclosed musculardystrophy-associated molecules indicates that the one or more testagents is of use to treat the muscular dystrophy.

In one example, determining whether there is differential expression ofone or more muscular dystrophy-associated molecules is by use of an invitro assay. For example, an in vitro assay can be employed to compareexpression of one or more muscular dystrophy-associated molecules in asample, such as a blood or urine sample, in the presence and absence ofthe test agent. Expression levels can be determined by methods known tothose of skill in the art including real time quantitative polymerasechain reaction, microarray analysis or Western blot analysis. In someexamples, an at least 2-fold, at least 3-fold, or at least 5-fold,decrease in the activity of one or more disclosed musculardystrophy-associated molecules in the presence of the one or more testagents as compared to the reference value indicates the one or more testagents is of use to treat muscular dystrophy. In some examples, an atleast 2-fold, at least 3-fold, or at least 5-fold, increase in theactivity of one or more disclosed muscular dystrophy-associatedmolecules in the presence of the one or more test agents as compared tothe reference value indicates the one or more test agents is of use totreat muscular dystrophy.

Test Agents

The one or more test agents can be any substance, including, but notlimited to, a protein (such as an antibody), a nucleic acid molecule(such as a siRNA), an organic compound, an inorganic compound, a smallmolecule or any other molecule of interest. In a particular example, thetest agent is a siRNA that reduces or inhibits the activity (such as theexpression) of one of the disclosed muscular dystrophy-associatedmolecules, such as disintegrin and metalloproteinase with thrombospondinmotifs 5 (Adamts5), agrin (Agrn), collagen 6A1 (Col6a1), Galectin-3,matrix metalloproteinase 2 (Mmp2), integrin α3 (Iga3), integrin α6(Iga6), integrin α7 (Iga7), laminin-α4 (Lama4), laminin-α5 (Lama5),nidogen 1 (Nid1), tenascin C (Tnc), tissue inhibitor ofmetalloproteinase 1 (Timp1), tissue inhibitor of metalloproteinase 2(Timp2). For example, the siRNA is directed to Galectin-3.

In other examples, the test agent is an antibody. For example, theantibody is directed to specifically bind to one of the disclosedmuscular dystrophy-associated molecules, such as disintegrin andmetalloproteinase with thrombospondin motifs 5 (Adamts5), agrin (Agrn),collagen 6A1 (Col6a1), Galectin-1, Galectin-3, matrix metalloproteinase2 (Mmp2), integrin α3 (Iga3), integrin α6 (Iga6), integrin α7 (Iga7),laminin-α4 (Lama4), laminin-α5 (Lama5), nidogen 1 (Nid1), tenascin C(Tnc), tissue inhibitor of metalloproteinase 1 (Timp1), tissue inhibitorof metalloproteinase 2 (Timp2). In a particular example, the antibody isdirected to Galectin-1 or Galectin-3.

Disclosed test agents also include aptamers, In one example, an aptameris a single stranded nucleic acid molecule (such as, DNA or RNA) thatassumes a specific, sequence dependent shape and binds to a targetprotein (e.g., Galectin-1 or Galectin-3) with high affinity andspecificity. Aptamers generally comprise fewer than 100 nucleotides,fewer than 75 nucleotides, or fewer than 50 nucleotides (such as 10 to95 nucleotides, 25 to 80 nucleotides, 30 to 75 nucleotides, or 25 to 50nucleotides). In a specific embodiment, a disclosed diagnostic specificbinding reagent is a mirror image aptamer (also called a SPIEGELMERTM).Mirror image aptamers are high affinity L enantiomeric nucleic acids(for example, L ribose or L 2′-deoxyribose units) that display highresistance to enzymatic degradation compared with D oligonucleotides(such as, aptamers). The target binding properties of aptamers andmirror image aptamers are designed by an in vitro selection processstarting from a random pool of oligonucleotides, as described forexample, in Wlotzka et al., Proc. Natl. Acad. Sci. 99(13):8898 8902,2002. Methods of generating aptamers are known in the art (see e.g.,Fitzwater and Polisky (Methods Enzymol., 267:275-301, 1996; Murphy etal., Nucl. Acids Res. 31:e110, 2003).

In another example, an aptamer is a peptide aptamer that binds to atarget protein (e.g., a Galectin-1 or Galectin-3) with high affinity andspecificity. Peptide aptamers can include a peptide loop (e.g., which isspecific for Galectin-1 or Galectin-3) attached at both ends to aprotein scaffold. This double structural constraint greatly increasesthe binding affinity of the peptide aptamer to levels comparable to anantibody's (nanomolar range). The variable loop length is typically 8 to20 amino acids (e.g., 8 to 12 amino acids), and the scaffold may be anyprotein which is stable, soluble, small, and non-toxic (e.g.,thioredoxin-A, stefin A triple mutant, green fluorescent protein, eglinC, and cellular transcription factor Sp1). Peptide aptamer selection canbe made using different systems, such as the yeast two-hybrid system(e.g., Gal4 yeast-two-hybrid system) or the LexA interaction trapsystem.

VII. Kits

Provided by this disclosure are kits that can be used to diagnose,prognose or treat muscular dystrophy. For example, a kit is disclosedherein for diagnosing or prognosing muscular dystrophy, such as DMD,LGMD, FHMD, BMD or MDC1A, by reducing or inhibiting one or more symptomsassociated with the muscular dystrophy in which the kit includes atleast one agent capable of altering the expression or biologicalactivity of one or more of the disclosed muscular dystrophy-associatedmolecules. The disclosed kits can include instructional materialsdisclosing means of use of the compositions in the kit. Theinstructional materials can be written, in an electronic form (such as acomputer diskette or compact disk) or can be visual (such as videofiles). For example, instructions indicate to first perform a baselinemeasurement of a particular activity, such as measuring expressionlevels of one or more of the disclosed muscular dystrophy-associatedmolecules, such as Galectin-1 or Galectin-3. Then, administer acomposition known to regulate such molecules according to the teachingsherein. Administration is followed by re-measuring the particularactivity. The activity level prior to treatment is compared to activityobserved following treatment. An alteration in activity of at least 10%,for example, about 15% to about 98%, about 30% to about 95%, about 40%to about 80%, about 50% to about 70%, including about 20%, about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about95%, about 98% or about 100%, as compared to such activity in theabsence of the composition indicates an effective treatment. Inparticular embodiments, a greater than 50% alteration indicates aneffective treatment. An effective treatment can include, but are notlimited to, an increase in patient survival, a slowing of theprogression of the particular type of muscular dystrophy, a goodprognosis, or a prevention of further muscle damage.

Kits are provided that can be used in the therapy assays disclosedherein. For example, kits can include one or more compositions, agents(such as antibodies) capable of detecting one or more of the musculardystrophy biomarkers (for example, measuring Galectin-1 or Galectin-3,or combinations thereof). One skilled in the art will appreciate thatthe kits can include other agents to facilitate the particularapplication for which the kit is designed.

In one example, a kit is provided for treating DMD. For example, suchkits can include one or more compositions capable of targetinginhibiting or reducing Galectin-3 activity or expression.

In some examples, a kit is provided for detecting one or more of thedisclosed muscular dystrophy biomarkers in a biological sample. Kits fordetecting muscular dystrophy-associated molecules can include one ormore probes that specifically bind to the molecules. In an example, akit includes an array with one or more of disintegrin andmetalloproteinase with thrombospondin motifs 5 (Adamts5), agrin (Agra),collagen 6A1 (Col6a1), Galectin-1, Galectin-3, matrix metalloproteinase2 (Mmp2), integrin α3 (Iga3), integrin α6 (Iga6), integrin α7 (Iga7),laminin-α4 (Lama4), laminin-α5 (Lama5), nidogen 1 (Nid1), tenascin C(Tnc), tissue inhibitor of metalloproteinase 1 (Timp1), tissue inhibitorof metalloproteinase 2 (Timp2) or any combination thereof and controls,such as positive and negative controls. In other examples, kits includeantibodies that specifically bind to one of the musculardystrophy-associated biomarkers disclosed herein. In some examples, theantibody is labeled (for example, with a fluorescent, radioactive, or anenzymatic label). Such a diagnostic kit can additionally contain meansof detecting a label (such as enzyme substrates for enzymatic labels,filter sets to detect fluorescent labels, appropriate secondary labelssuch as a secondary antibody, or the like), as well as buffers and otherreagents routinely used for the practice of a particular diagnosticmethod. In some examples, a kit includes at least one probe or antibodythat specifically binds to Galectin-3 and the kit is used to diagnose orprognose DMD, LGMD, FHMD, BMD or MDC1A. In some examples, a kit includesat least one probe or antibody that specifically binds to Galectin-3 andthe kit is used to prognose DMD and/or determine the efficacy of atreatment for DMD.

The disclosure is further illustrated by the following non-limitingExamples.

EXAMPLES Example 1 Biomarkers for MDC1A

This example investigates the use of Galectin-1 and Galectin-3 abiomarkers for MDC1A.

i. Materials and Methods

Western blotting. Gastrocnemius muscles from 4- and 8-week old malewild-type and dy^(W) −/− animals were pulverized with a mortar andpestle cooled in liquid nitrogen. Protein was extracted from both serumand muscle tissue in RIPA buffer (50 mM Hepes pH 7.4, 150 mM NaCl, 1 mMNa₃VO₄, 10 mM NaF, 0.5% Triton X-100, 0.5% NP50, 10% glycerol, 2 mM PMSFand a 1:200 dilution of Protease Inhibitor Cocktail Set III) andquantified using a Bradford assay (Bio-Rad Laboratories Inc, Herculues,Calif.). Proteins were separated by SDS-PAGE. Galectin-1 was detectedusing a 1:1000 dilution of anti-Galectin-1 antibody (H00003956-D01PAbnova, Walnut, Calif.). Galectin-3 was detected using a 1:1000 dilutionof anti-galecin-3 antibody (ab53082, Abcam). Blots were incubated withprimary antibody overnight at 4° C. Blots were then incubated with a1:5000 dilution of goat-anti-rabbit-IgG secondary antibody (Li-CorBiosciences, Lincoln, Nebr.) for 1 hour. Blots were imaged using anOdyssey Imaging System and bands were quantified using the same system.Tissue blots were normalized to a-tubulin using a 1:5000 dilution ofanti-α-tubulin (AbCam, Cambridge, Mass.) followed by agoat-anti-mouse-IgG (Li-Cor Biosciences, Lincoln, Nebr.).

Immunofluorescence. Cryosections (8 mm) of 4- and 8-week old maletibialis anterior (TA) muscles were cut using a LeicaCM 1850 cryostatand mounted onto pre-cleaned Surgipath slides. Sections were fixed using4% paraformaldehyde (PFA) for 5 minutes then rehydrated using PBS.Slides were blocked in 5% BSA in PBS then incubated with a 1:500dilution of ab53082 (AbCam) for 1 hour. Slides were then incubated witha 1:1000 dilution of FITC-conjugated anti-rabbit-IgG antibody for 1hour. Slides were mounted using Vectashield with DAPI and imaged using aZeiss Axioskop 2 plus fluorescence microscope. Images were capturedusing a Zeiss AxioCam HRc digital camera with Axiovision 4.1 software.

Quantitative real-time PCR analysis. Total RNA was purified from five 4-and 8-week old male wild-type and dy^(W) −/− grastrocnemius musclesusing Trizol (Invitrogen, Carlsbad, Calif.) reagent. After theconcentration was determined, mRNA was pooled equally by genotype forcDNA production. The cDNA was prepared from 3 μg of pooled total RNAwith random hexamers and Superscript III (Invitrogen, Carlsbad, Calif.)using standard procedures. Quantitative real-time PCR was conducted with50 pg total cDNA using SYBR Green Jumpstart (Sigma-Aldrich, St Louis,Mo.) with Lgals1 primer sequences and Lgals3 primer sequences and levelswere normalized to that of Gapdh.

Statistics. The fold change over wild-type was calculated using the ΔΔCtmethod after normalization and the average fold change in transcript and(±s.e.m.) were calculated. One and two way ANOVA with a Bonferroni posttest correction were used to determine statistical significance usingGraphPad Prism

ii. Results

Quantitative Real-Time PCR was used to determine changes in thetranscription of Lgals1 and Lgals3 (FIGS. 1A-1B). Both 4- and 8-week olddystrophic mice had significantly increased transcripts of Lgals3compared to age-matched wild-type mice. The 4-week old mice had 70.02fold increase in Galectin-3 transcript compared to wild-type animals.Lgals3 transcription was reduced in the 8-week old mice; however, it wasstill significantly elevated 9.37 fold compared to wild-type animals(FIG. 1B). These results indicate the loss of laminin-α2 resulted inincreased transcription of Galectin-3 and that transcription levels dropas the dystrophic mice age.

Both 4- and 8-week old dystrophic mice had significantly increasedtranscripts of Lgals1 compared to age-matched wild-type animals as well.The 4-week old mice had a 9.19 fold increase in Galectin-1 transcriptcompared to wild-type animals. Lgals1 transcription was reduced in the8-week old mice; however, it was still significantly elevated 1.7 foldcompared to wild-type animals (FIG. 1A). These results indicate the lossof laminin-α2 results in increased transcription of Galectin-1 and thattranscription levels drop as the dystrophic mice age.

Western blotting analysis revealed no significant difference inGalectin-1 protein levels in 4- or 8-week old dy^(W) −/− animals whencompared to age-matched wild-type animals (FIG. 2A and 2B,respectively). There was also no significant difference between theGalectin-1 protein when comparing 4- and 8-week old dy^(W) −/− animals(FIG. 2C).

Western blotting analysis for Galectin-3 protein revealed significantlymore Galectin-3 protein in 4-week old dy^(W) −/− animals compared toage-matched wild-type animals (FIGS. 3A and 3B) and 8-week old dy^(W)−/− animals compared to age-matched wild-type animals.

Western blotting on serum revealed no significant difference inGalectin-3 protein between 4-week old dy^(W) −/− mice and age-matchedwild-type mice (FIG. 4A). In addition, serum western blots showedsignificantly more Galectin-3 protein in 4-week old dy^(W) −/− mice than8-week old dy^(W) −/− mice (FIG. 4B). These results revealed that theamount of Galectin-3 released into the blood stream is different thanthat held in the muscle.

Immunofluorescence for Galectin-3 was also completed on the tibialisanterior muscle of 4- and 8-week old dy^(W) −/− mice and wild-type mice.Immunofluorescence revealed a similar pattern as to that shown in thetissue western blots. 4-week old dy^(W) −/− mice had elevated levels ofGalectin-3 compared to age-matched wild-type animals. Galectin-3 levelsappeared to be similar in 4- and 8-week old dy^(W) −/− mice as well asbetween 8-week old dy^(W) −/− and age-matched wild-type mice. Galectin-3levels also appeared to increase as the wild-type animals age (FIG. 5).

Although Galectin-1 transcript was significantly elevated in the dy^(W)−/− animals, this did not translate to an elevation in detectableGalectin-1 protein. These studies indicate that Galectin-1 is not a goodcandidate as a biomarker for the dy^(W) −/− mouse model of MDC1A. Incontrast, Galectin-3 was significantly elevated at the transcript levelof dy^(W) −/− mice, and at the protein level in the muscle, indicatingits use as a biomarker of MDC1A.

Example 2 Biomarkers for DMD

This example demonstrates the use of Galectin-1 and Galectin-3 abiomarkers for DMD.

i. Materials and Methods

Western blotting. Gastrocnemius muscles from 2-, 5- and 10-week old malewild-type and mdx animals were pulverized with a mortar and pestlecooled in liquid nitrogen. Protein was extracted from both serum andmuscle tissue in RIPA buffer (50 mM Hepes pH 7.4, 150 mM NaCl, 1 mMNa₃VO₄, 10 mM NaF, 0.5% Triton X-100, 0.5% NP50, 10% glycerol, 2 mM PMSFand a 1:200 dilution of Protease Inhibitor Cocktail Set III) andquantified using a Bradford assay (Bio-Rad Laboratories Inc, Herculues,Calif.). Proteins were separated by SDS-PAGE. Galectin-1 was detectedusing a 1:1000 dilution of anti-Galectin-1 antibody (H00003956-D01PAbnova, Walnut, Calif.). Galectin-3 was detected using a 1:1000 dilutionof anti-galecin-3 antibody (ab53082, Abcam). Blots were incubated withprimary antibody overnight at 4° C. Blots were then incubated with a1:5000 dilution of goat-anti-rabbit-IgG secondary antibody (Li-CorBiosciences, Lincoln, Nebr.) for 1 hour. Blots were imaged using anOdyssey Imaging System and bands were quantified using the same system.Blots were normalized to a-tubulin using a 1:5000 dilution ofanti-α-tubulin (AbCam, Cambridge, Mass.) followed by agoat-anti-mouse-IgG (Li-Cor Biosciences, Lincol, Nebr.).

Immunofluorescence. Cryosections (8 mm) of 5- and 10-week old maletibialis anterior (TA) muscles were cut using a LeicaCM 1850 cryostatand mounted onto pre-cleaned Surgipath slides. Sections were fixed using4% paraformaldehyde (PFA) for 5 minutes then rehydrated suing PBS.Slides were blocked in 5% BSA in PBS then incubated with a 1:500dilution of ab53082 (AbCam) for 1 hour. Slides were then incubated witha 1:1000 dilution of FITC-conjugated anti-rabbit-IgG antibody for 1hour. Slides were mounted using Vectashield with DAPI and imaged using aZeiss Axioskop 2 plus fluorescence microscope. Images were capturedusing a Zeiss AxioCam HRc digital camera with Axiovision 4.1 software.

Quantitative real-time PCR analysis. Total RNA was purified from five 5-and 10-week old male wild-type and mdx grastrocnemius muscles usingTrizol (Invitrogen, Carlsbad, Calif.) reagent. After the concentrationwas determined, mRNA was pooled equally by genotype for cDNA production.The cDNA was prepared from 3 μg of pooled total RNA with random hexamersand Superscript HI (Invitrogen, Carlsbad, Calif.) using standardprocedures. Quantitative real-time PCR was conducted with 50 pg totalcDNA using SYBR Green Jumpstart (Sigma-Aldrich, St Louis, Mo.) withLgals1 primer sequences and Lgals3 primer sequences (and levels werenormalized to that of Gapdh.

Statistics. The fold change over wild-type was calculated using the ΔΔCtmethod after normalization and the average fold change in transcript and(±s.e.m.) were calculated. One and two way ANOVA with a Bonferroni posttest correction were used to determine statistical significance usingGraphPad Prism.

ii. Results

Quantitative Real-Time-PCR was used to determine changes in thetranscription of Lgals1 and Lgals3. Both 5- and 10-week old dystrophicmice had significantly increased transcripts of Lgals3 compared toage-matched wild-type mice. The 5-week old mice had an 11.42 foldincrease in Galectin-3 transcript compared to wild-type animals, whilethe 10-week old mice had a 67.20 fold increase in Galectin-3 transcriptcompared to wild-type animals. Transcript levels of Lgals3 alsoincreased from the 5-week old mdx mice (11.42 fold increase) to the10-week old mdx mice (67.20 fold increase). These results indicate theloss of dystrophin resulted in increased transcription of Galectin-3 andthat transcription levels increased as the dystrophic mice age.

Only the 10-week old mdx mice had significantly increased levels ofLgals1 transcript compared to wild-type animals. The 5-week old mice hada 1.49 fold increase in Galectin-1 transcript, while the 10-week olddystrophic mice had a 2.51 fold increase in Galectin-1 transcriptcompared to wild-type animals. In addition, transcript levels of Lgals1increased from the 5-week old mdx mice (1.49 fold increase) to the10-week old mdx mice (2.51 fold increase) (FIG. 6). These resultsindicate the loss of dytstrophin resulted in increased transcription ofGalectin-1 and that transcription levels increased as the dystrophicmice age.

Western blotting analysis revealed no significant difference inGalectin-1 protein levels in the gastrocnemius muscle of 5- or 10-weekold mdx animals when compared to age-matched wild-type animals (FIG. 7Aand 7B, respectively). There was, however, a significant differencebetween the Galectin-1 protein when comparing 2-, 5- and 10-week old mdxanimals (FIG. 7C).

At 5-weeks of age, the mdx animals had significantly more Galectin-3protein in the gastrocnemius muscle than the wild-type animals (FIG.8A.). In addition, there was a significant difference in the Galectin-3protein levels between 5- and 10-week old mdx animals (FIG. 8B).

Western blotting on 5- and 10-week old mdx and wild-type serum revealedsimilar results to the tissue blots, although there was no significantdifference in Galectin-3 protein levels (FIG. 9A and FIG. 9B,respectively). However, at both age points the mdx mice were trendingtowards more Galectin-3 protein.

Immunofluorescence for Galectin-3 was also completed on the tibialisanterior (TA) muscle of 5- and 10-week old mdx and wild-type mice.Immunofluorescence revealed a similar pattern as to that shown throughwestern blotting. Both 5- and 10-week old mdx mice had elevated levelsof Galectin-3 compared to age-matched wild-type animals. Galectin-3levels also appeared to be elevated in 10-week old mdx mice compared to5-week old mdx mice (FIG. 10).

These results revealed that although Galectin-1 transcript wassignificantly elevated in the mdx animals, is did not translate to anelevation in detectable Galectin-1 protein. Western blotting revealed nosignificant difference between Galectin-1 levels in mdx and wild-typemice, but a significant difference was seen between 5- and 10-week oldmdx mice. A biomarker needs to not only follow the progression of thedisease, but must also be differentiable from levels found indisease-free patients. As the results do anot reveal these differencesin Galectin-1 levels, it is not a good candidate as a biomarker for themdx mouse model of DMD.

In this study, however, Galectin-3 was significantly elevated at thetranscript level of mdx mice, as well as at the protein level in themuscle. Galectin-3 was secreted by macrophages and monocytes, two cellsseen in fibrosis, a hallmark of DMD. Therefore, these studies supportthe use of Galectin-3 as a biomarker for DMD.

Additional studies have been performed evaluating the levels ofGalectin-3 in the muscle of the GRMD dog model of DMD. The GRMD modeldevelops progressive and fatal muscle disease and has been shown toexhibit pathophysiological disease features identical to DMD includingprogressive loss of muscle function, muscle membrane fragility,cardiomyopathy and premature death (Kornegay et al., Muscle Nerve11:1056-1064, 1988; Cooper et al., Nature 334:154-156, 1988). The GRMDdog model is generally accepted as the gold standard preclinical modelto test therapeutics for DMD.

Western analysis revealed GRMD dogs had increased Galectin-3 levels inthe Vastus lateralus muscle of GRMD as compared to control dogs (FIG.11). Immunofluorescence studies revealed that Galectin-3 is foundsurrounding myofibers and associated with blood vessels of unaffecteddogs. In sharp contrast, the amount of Galectin-3 increases in muscleand localization changes to discrete sites that are associated withsmooth muscle actin positive regions which correspond to small bloodvessels. In unaffected dogs, Galectin-3 was localized around myofibersand associated with large blood vessels within the endothelial/smoothmuscle of such vessels. Loss of dystrophin in GRMD dogs resulted inhigher levels of Galectin-3 with punctate staining in skeletal muscletissue. There was also a loss of Galectin-3 localization from largemuscle blood vessels to smaller blood vessels in GRMD dogs and thecolocalization of Galectin-3 and smooth muscle actin was lost.

Galectin3 serum levels in wild-type and mdx mice were determined byELISA (Table 3). There was very little variation in the wild-type (WT)control animals at all ages observed. Mdx animals exhibited a steadyincrease in average serum levels from 5 weeks to 10 weeks, whendystrophic pathology were most consistently observed. After 10 weeksthere was variance in these animals. Exercise had no effect on the serumlevels of 5-week old mdx mice.

TABLE 3 Galectin3 serum levels in wild-type and mdx mice. Age AverageSerum Level (months) (ng/mL) SEM N WT 3.5 47.76 5.71 3 C57BL/10 9.540.92 4.67 8 10 52.09 5.62 7 11 54.90 23.56 4 12 50.97 5.70 10 mdx 1.2545.40 11.05 8 2 108.45 29.96 5 3 120.11 21.04 15 4.5 47.86 18.00 2 586.55 45.58 2 5.5 71.16 17.47 7 6 301.90 153.33 5 7.5 557.53 198.73 48.5 141.38 76.98 3 9 204.76 85.47 10 9.5 207.63 176.75 2 exercised 1.2560.14 10.48 4 mdx

In addition, Galectin-3 serum levels were measured in MDC1A patients andcompared to age matched controls (Table 4). Patients and controls werebroken into age-matched categories by age or gender starting with up tothree years in which patients had significantly higher serum levels thencontrols. After age 3, for both males and females, patients appear tohave slightly lower serum levels. A final category for patients wasbased on a lack of muscular ability and these patients had the lowestserum levels. Table 5 provides a summary of the MDC1A patient data shownin Table 4.

TABLE 4 Galectin-3 serum analysis of MDC1A patients compared to agematched controls. Galectin- Muscular 3 pg/mL Age Gender AbilitiesControls 1845 0.9 M 3091 1.3 M 4867 1.5 F 4193 2.5 F 3441 2.7 M 2357 4.0F 5825 5.9 M 6288 6.4 M 2185 7.3 F 5412 7.9 M Patients 5687 0.9 M goodhead control, unable to sit without assistance 1172 1.4 M briefly ableto sit without assistance 7367 1.5 F sat without assistance 12263 1.7 Fsat without assistance 5002 2.8 M sat without assistance 1549 3.9 F satwithout assistance 4372 5.6 M sat without assistance 4417 6.5 M satwithout assistance 3135 7.8 M sat without assistance 633 11.6 F goodhead control, unable to sit without assistance

TABLE 5 Average Serum Levels of Galectin-3 in various patientpopulations. Average Serum Levels (ng/mL) N Controls Under 3 3.49 5 (3M,2F) Patients Under 3 (mobile) 7.58 4 (2M, 2F) Patient Under 3(non-mobile) 1.17 1M Male Controls Over 3 5.84 3 Male Patients Over 3(mobile) 3.97 3 Female Controls Over 3 2.27 2 Female Patient Over 3(mobile) 1.55 1 Female Patient Over 3 (non-mobile) 0.63 1

The dyw −/− mouse has a bell shaped curve for Galectin-3 protein inmuscle which peaks around 4 weeks and then falls by 8 weeks as the micebecame less and less active due to muscle weakness. A similar patternwas observed in MDC1A patient serum compared to controls. Before the ageof 3, the average serum level of Galectin-3 was over 2-fold higher thanthat in controls (˜7.5 ng/mL compared to 3.5 ng/mL, respectively).However, after the age of 3 the levels were lower than gender matchedcontrols. The observed decrease indicates a significant loss of muscleas supported by the fact that the two patients who were non-mobile andunable to sit without assistance had extremely low serum levels ofGalectin-3. These studies further indicate a role of Galectin-3 in MDand a use of such to indicate the presence of MD. Additionally, forMDC1A, Galectin-3 serum levels are diagnostic before the age of 3 (highGal-3 serum levels) and potentially prognostic for severe pathology(extremely low Gal-3 serum levels).

Example 3 Additional Biomarkers for MDC1A

This example describes possible biomarkers for MDC1A.

i. Material and Methods

Transgenic α7 integrin dy^(W) −/− mice. Transgenic α7 integrin dy^(W)−/− mice were generated by breeding mice that overexpressed the α7BX2integrin in skeletal muscle with dy^(W) +/− animals. Resultant pupswhich were heterozygous for the laminin α2 mutant allele and positivefor the α7 BX2 transgene were bred to dy^(W) +/− mice. The male pupsfrom these matings included dy^(W) +/+; itga7-(wild-type), dy^(W) −/−;itga7-(dy^(W) −/−) (laminin-α2 deficient) and dy^(W) −/−; itga7+(laminin-α2 deficient that overexpress the α7 BX2 integrin) mice. Malelittermates were used as controls for all studies. Genomic DNA wasisolated from tail biopsies taken at 10 days of age using the Wizard SVGenomic DNA Purification System (Promega, Madison, Wis.). Polymerasechain reaction (PCR) was used as previously described to detect thelaminin-α2 allele and the α7 BX2 transgene.

Isolation of Skeletal Muscle. Four-week-old wild-type, dy^(W) −/− anddy^(W) −/−; itga 7+ male mice were sacrificed, Skeletal muscles weredissected and flash frozen in liquid nitrogen cooled isopentane. Tissueswere stored at −80° C.

Western blot analysis. Gastrocnemius muscles from 4 week old male micewere pulverized with a mortar and pestle cooled in liquid nitrogen.Protein was extracted in RIPA buffer (50 mM Hepes pH 7.4, 150 mM NaCl, 1mM Na₃VO₄, 10 mM NaF, 0.5% Triton X-100, 0.5% NP50, 10% glycerol, 2 mMPMSF and a 1:200 dilution of Protease Inhibitor Cocktail Set III) andquantified by a Bradford assay (Bio-Rad Laboratories Inc, Hercules,Calif.). Proteins were separated by SDS-PAGE. The α7 integrin wasdetected with a 1:1000 dilution of anti-α7B antibody overnight. Integrinα7A was detected using 1:1000 dilution of CDB 345 antibody overnight.Integrin α3 was quantified using AB1920 antibody (Chemicon). The α1Dintegrin was visualized using α1D-antibody overnight. All primaryantibodies were followed by a 1:5000 goat-anti-rabbit secondary antibody(Li-Cor Biosciences, Lincoln, Nebr.) for 1 hour . Galectin-1 wasdetected with a 1:1000 dilution of H00003956-D01P (Abnova, Walnut,Calif.). Galectin-3 was detected with a 1:1000 dilution of ab53082(Abeam, Cambridge, Mass.). Immunoblots were normalized by using a 1:5000dilution of an anti-a tubulin (AbCam, Cambridge, Mass.) antibodyfollowed by a 1:5000 dilution of goat-anti-mouse secondary antibody,Band intensities were determined with an Odyssey Imaging System.

Immunofluorescence. Cryosections (8 μm) of 4 week old male TibialisAnterior (TA) muscle were cut using a LeicaCM 1850 cryostat and mountedon precleaned Surgipath slides. Sections were fixed using 4%paraformaldehyde (PFA) for 2 or 5 minutes then rehydrated usingPhosphate Buffered Saline (PBS). Slides were blocked in 5% Bovine SerumAlbumin (BSA) in PBS then incubated with laminin-α2G or α1D integrinantibody. For detection of Galectin-1 H00003959-D01P (Abnova) antibodywas used. Galectin-3 was visualized using ab53082 (abcam). AntibodyT3413 (Sigma) was used to detect Tenascin C. MMP2 and TIMP1 weredetected using antibodies ab37150 and ab86482 respectively (Abcam).Slides were then incubated using an appropriate secondary which was aFITC-anti rabbit in all cases except for Tenascin C which was aFITC-anti rat secondary antibody. For detection of spectrin, slides werefixed for 1 minute in ice cold acetone then treated with the M.O.M™ kitaccording to package instructions (FMK-2201 Vector Laboratories, inc.Burlingame, Calif.). A mouse monoclonal spectrin antibody (Novo CastraNCL-spec2) was then used at 1:100 for 30 minutes followed by a FITC-antimouse secondary at 1:1000 for 1 hour. Slides were mounted usingVectashield with DAPI and imaged using a Zeiss Axioskop 2 Plusfluorescent microscope. Images were captured using a Zeiss AxioCam HRcdigital camera with Axiovision 4.1 software.

Inflammatory Cell Infiltrate. Four week old TA muscle cryosections werefixed in 4% PFA for 5 minutes followed by rehydration with PBS. Slideswere incubated with FITC Rat Anti-Mouse CD11b antibody (BD Biosciences,San Jose, Calif.) at 1:1000 for 1 hour to detect macrophages in themuscle tissue. Slides were washed with PBS and mounted using Vectashieldwith DAPI. Muscle sections from five mice of each genotype were analyzedand CD11b positive cells per twenty fields at 400× magnification werecounted. A Zeiss Axioskop 2 Plus fluorescent microscope was used to viewthe slides and images were captured using a Zeiss AxioCam HRc digitalcamera with Axiovision 4.1 software

Confocal microscopy. The TA muscles from 4 week old male mice from eachgenotype were sectioned and subjected to immunfluorescence. Fordetection of α7B integrin sections were fixed in ice cold acetone (−20°C.) for 1 minute then rehydrated using phosphate-buffered saline (PBS).Cryosections were blocked in a 5% bovine serum albumin in PBS solutionfor 20 minutes followed by incubation with CDB347 (which recognizes thecytoplasmic domain of both mouse and rat α7B integrin) or α1D A2antibodies for 1 hour. Slides were then washed with 1% BSA and incubatedwith FITC-conjugated anti-rabbit antibody for 1 hour. Slides were againwashed with 1% BSA. To outline the myofibers sections were incubatedwith rhodamine labeled wheat germ agglutinin for 30 minutes. Slides weremounted using Vectashield with DAPI. Images were captured using anOlympus Fluoview™ Confocal Scanning System.

Survival and weight gain analysis. Male mice were allowed to age andmonitored daily for weight loss and any signs of pain, distress orillness. A weight loss of >10% over a one week period was alsoconsidered a terminal sign and the animals were humanely euthanized.Weights from animals of each genotype were compared at 3, 8, and 12weeks of age.

Grip strength and activity assays. The forelimb grip strength of fourand eight week-old male wild-type, dy^(W) −/− and dy^(W) −/−; itga7+mice were measured using a SDI Grip Strength System and a Chatillon DFEDigital Force Gauge (San Diego Instruments, Inc., San Diego, Calif.) asper standard protocol. Five consecutive tests were performed for eachmouse and the data averaged for each mouse genotype. In order to assessmobility four and eight week old male wild-type, dy^(W) −/− and dy^(W)−/−; itga7+ mice were placed in a clean cage by themselves and monitoredfor five minutes. Periods of moving about the cage, standing up, anddigging were considered times of activity. Additionally during this timeperiod the number of times the mouse stood up was recorded. Stand uptesting was only performed on animals which were physically able tostand up. Some mice were excluded from these samples due to the extentof their peripheral neuropathy.

Hematoxylin and Eosin Staining. Cryosections from 4 week-old TA anddiaphragm muscle were stained using Hematoxylin and Eosin and used todetermine the percentage of myofibers that contained centrally locatednuclei using a Zeiss Axioskop 2 plus fluorescent microscope. A minimumof 1000 fibers per animal (5 animals per group) were counted and thepercentage of myofibers with centrally located nuclei calculated. Imageswere captured using a Zeiss AxioCam HRc digital camera and Axiovision4.1 software.

Myofiber Area Determination. Cryosections from 4 week old TA anddiaphragm muscles were fixed for 5 minutes in 4% paraformaldehyde (PFA)and rehydrated in PBS. Myofibers were outlined with 2 μg/ml OregonGreen-488 conjugated WGA (Molecular Bioprobes, Eugene, Oreg.) for 30minutes. Sections were then washed with PBS for 15 minutes and mountedin Vectashield. A minimum of 1000 fibers per animal with five animalsper group were assessed for the TA muscle. For diaphragm muscle aminimum of 500 fibers per animal with five animals per genotyped wereused. Myofiber cross-sectional area was determined with a Zeiss Axioskop2 Plus fluorescent microscope and images were captured with a ZeissAxioCam HRc digital camera with Axiovision 4.1 software.

Quantitative real-time PCR analysis. Total RNA was purified from five 4week old male mice wild-type, dy^(W) −/−, and dy^(W) −/−; itga7+gastrocnemius muscles using Trizol (Invitrogen, Carlsbad, CA) reagent.After the concentration was determined, mRNA was pooled equally bygenotype for cDNA production. The cDNA was prepared from 4 μg of pooledtotal RNA with random hexamers and Superscript III (Invitrogen,Carlsbad, CA) using standard procedures. Quantitative real-time PCR wasconducted with 50 pg total cDNA using SYBR Green Jumpstart(Sigma-Aldrich, St Louis, Mo.) with primer sequences to mouseextracellular matrix genes are listed in Table 2 and normalized toGapdh. The fold change over wild-type was calculated using the ΔΔCtmethod after normalization and the average fold change in transcript andstandard error of the mean were calculated.

Statistics. Data is reported as the mean +/− standard deviation. One wayanalysis of variance (ANOVA) was used to compare animals across groups.Kaplan-Meier Log-Rank test was used to determine significance of lifespan changes. Myofiber cross-sectional area was analyzed using theGLIMMIX statistical analysis package in SAS. A p-value of <0.05 wasconsidered significant.

ii. Results

Transgenic α7 integrin expression alters the composition of theextracellular matrix in laminin-α2 deficient muscle.

The loss of laminin-211/221 in the muscle extracellular matrix is anunderlying cause of muscle disease in MDC1A. Since the α7 integrin is amajor laminin receptor in muscle we next determined the mechanism bywhich increased α7β1 integrin rescued dy^(W) −/− mice in the absence ofits laminin-211/221 ligand. QRT-PCR was used to examine the expressionprofile of genes encoding an array of extracellular matrix proteins inthe gastrocnemius muscle of 4 week old wild-type, dy^(W) −/− and dy^(W)−/−; itga7+ mice. QRT-PCR revealed that dy^(W) −/− mice exhibitedincreased levels of a disintegrin and metalloproteinase withthrombospondin motifs 5 (Adamts5), agrin (Agn), collagen 6A1 (Col6A1),Galectin-1 (Lgals1), Galectin-3 (Lgals3), matrix metalloprotease 2(Mmp2), integrin α3 (Itga3), Integrin α6 (Itgα6), Integrin α7 (Itga7),laminin-α4 (Lama4), laminin-α5 (Lama5), nidogen (Nid1), tenascin C(TnC), tissue inhibitor of metalloproteinase 1 (Timp1) and tissueinhibitor of metalloproteinase 2 (Timp2) transcripts compared towild-type (Table 6).

TABLE 6 Changes in gene expression in dy^(W)−/− mice. dy^(W)−/−dy^(W)−/−; itga7+ Significant Change (fold increase (fold increase(dy^(W)−/− vs dy^(W)−/−; Gene over over itga7+) Name Wild-type)Wild-type) (p-value < 0.05) Adamts5 1.97 ± 0.14 2.33 ± 0.08 No Agrn 9.23± 0.53 6.55 ± 0.15 Yes Col6a1 5.56 ± 0.27 7.45 ± 0.51 Yes Lgals1 9.19 ±0.28 12.13 ± 0.31  Yes Lgala3 70.02 ± 0.83  80.43 ± 1.96  Yes Mmp2 19.21± 0.86  12.40 ± 0.43  Yes Itga3 4.99 ± 0.41 4.53 ± 0.23 No Itga6 2.68 ±0.09 3.71 ± 0.09 Yes Itga7 4.08 ± 0.11 17.15 ± 0.42  Yes Lama4 11.96 ±0.40  12.60 ± 0.90  No Lama5 5.63 ± 0.34 6.16 ± 0.34 No Nid1 4.32 ± 1.566.07 ± 1.33 No Tnc 28.05 ±1.30  49.60 ± 3.64  Yes Timp1 276.20 ± 22.35 328.56 ± 20.40  Yes Timp2 6.30 ± 0.18 6.34 ± 0.21 No Results are thefold increase in expression compared with that in wild-type mice.Significance is taken as P < 5.05.

Transgenic expression of the α7 integrin in dy^(W) −/−; itga7+ miceresulted in reduced levels of agrin and Mmp2 transcripts compared tody^(W) −/− mice (Table 1). Transgenic expression of the α7 integrin indy^(W) −/−; itga7+ mice resulted in increased transcripts for CoI6A1,Lgals1, Lgals3, Itga3, Itga6, Itga7, Tnc and Timp1 compared to dy^(W)−/− mice (Table 1).

Next determined was if transgenic expression of the α7 integrin alteredexpression of Galectin-1 and -3 in the muscle of laminin-α2 null mice.Compared to wild-type mice, Galectin-1 transcript was increased 9.2-foldin dy^(W) −/− muscle and 12.1-fold in dy^(W) −/−; itga7+ animals (Table1). This increase in Galectin-1 transcript correlated with a 1.8-foldincrease in Galectin-1 protein in dy^(W) −/−; itga7+ animals compared towild-type. These results indicate an increase in Galectin-1 protein inthe gastrocnemius muscle of dy^(W) −/−; itga7+ animals.

Galectin-3 transcript was increased 70-fold and 80-fold in 4 week olddy^(W) −/− and dy^(W) −/−; itga7+ muscle respectively compared towild-type (Table 1). This increase in Galectin-3 transcript resulted ina 2-fold increase in Galectin-3 protein in dy^(W) −/− mice and a 7-foldincrease in Galectin-3 protein in dy^(W)−/−; itga7+ animals compared towild-type. These results indicate loss of laminin-α2 resulted inincreased Galectin-3 in the muscle extracellular matrix of dy^(W) −/−mice and that transgenic expression of α7 integrin further enhanced thelevels of Galectin-3 in laminin-α2 deficient muscle.

Tenascin C is normally localized at the myotendinous junctions and hasbeen shown to be enriched at extrajunctional sites of laminin-α2deficient muscle which correlate with regions of muscle regeneration.QRT-PCR was used to examine if transgenic overexpression of the α7integrin altered the expression of tenascin C in the muscle of dy^(W)−/− mice. QRT-PCR confirmed a 28-fold increase in tenascin C transcriptin the gastrocnemius muscle of dy^(W) −/− mice and a 49-fold increase intenascin C transcript in dy^(W) −/−; itga7+ gastrocnemius musclecompared to wild-type. These results indicate transgenic expression ofthe α7 integrin augmented tenascin C transcription in laminin-α2 nullmuscle.

Immunofluorescence was used to confirm qRT-PCR and immunoblotting forseveral proteins Immunofluorescence also demonstrated increasedextracellular galectin 1, Galectin-3, and Tenascin C in theextracellular matrix with Galectin-3 and tenascin C being more prevalentin the dy^(W) −/−; itga7+ mice. Immunostaining demonstrated reduced MMP2and increased TIMP1 in the extracellular matrix of the dy^(W) −/−;itga7+ mice compared with the dy^(W) −/− mice. These results indicatethat overexpression of the α7 integrin results in both augmentation andstabilization of the existing extracellular matrix in dy^(W) −/−; itga7+animals.

Transgenic expression of α7 integrin prevents muscle disease progressionin the diaphragm of dy^(W) mice.

MDC1A patients exhibit severe restrictive respiratory syndrome andrequire ventilator assistance to breathe as a result of severe diaphragmmuscle pathology. Histological analysis and measurements of myofiberarea were used to examine if transgenic expression of the α7 integrinprevented the onset of severe diaphragm muscle pathology. H&E studiesrevealed transgenic expression of the α7 integrin in 4 week old dy^(W)−/− diaphragm muscle resulted in reduced mononuclear cell infiltrate,hypotrophic muscle fibers, centrally located nuclei and fibrosis.

Analysis of myofiber cross-sectional areas confirmed the improvement inthe muscle pathology observed in the histological studies. Compared towild-type with a peak myofiber cross-sectional area of between 3.5-4.5μm², dy^(W) −/− muscle exhibited a large number of hypotrophic musclefibers with a peak myofiber area of only 2 μm². In contrast dy^(W) −/− ;itga7+ diaphragm myofibers exhibited a peak myofiber area of between3.5-5 μm² and a curve more similar to wild-type. At the maximumfrequency myofiber area, all three groups were significantly differentfrom one another. These results indicate transgenic expression of the α7integrin prevents muscle disease progression in the diaphragm oflaminin-α2 null mice. The studies described in this Example weredescribed in detail by Doe et al. in J. Cell Science: 124: 2287-2297,2011 which is hereby incorporated by reference in its entirety.

Example 4 Galectin-1 Treatment Decreases Muscle Damage in Mdx Mice

This example illustrates Galectin-1 increases muscle repair in mdx mice.

To produce recombinant Galectin-1, PCR amplified LGALS1 cDNA isolatedfrom total mouse muscle mRNA was cloned into a pET23b vector. Rosetta E.coli cells were transformed with pET23b-LGALS1 vector utilizing standardtechniques. Recombinant Galectin-1 was isolated and determined to have asequence corresponding to GENBANK® Accession No. NP_(—)032521.1 asprovided by GENBANK® on Aug. 10, 2012 except with a single amino acidsubstitution at amino acid position 10, in which glutamine (Q) wassubstituted for leucine (L). Recombinant Galectin-1 was purified byloading induced cell lysate onto Talon affinity column. The purity ofGalectin-1 fractions was then determined by using BCA protein analysis,Western blot analysis and Coomassie blue staining (see FIG. 12).

To determine the effect of Galectin-1 treatment on muscle damage in mdxmice, mdx mice were injected with 100 μl of 13 μM recombinant Galectin-1through intramuscular injections into their TA muscle. Sections of TAmuscle were stained using Hematoxylin and Eosin (H&E). The mdx TAinjected with Galectin-1 showed decreased muscle damage over thoseinjected with PBS, as indicated by decreased percentage of myofiberswith CLN (FIG. 13). FIG. 14 illustrates Galectin-1 treatment increasesα7 integrin. These studies indicate that Galectin-⅓ protein therapy canbe beneficial for MD- Galectin-1 increases alpha7 integrin and providesadditional extracellular matrix (ECM) for attachment of muscle cells.

Example 5 Galectin-1 Treatment Increases/Maintains Muscle Strengthand/or Bone Density

This example illustrates Galectin-1 treatment can be used to increaseand/or maintain muscle strength and/or bone density.

i. Material and Methods

Recombinant Galectin-1 Production. The mouse Galectin-1 cDNA wasproduced using standard reverse transcriptase (Superscript III,Invitrogen) from mouse muscle Total RNA (Trizol, Invitrogen) followed byPCR using Platinum Taq Supermix (Invitrogen). This PCR product was thensubcloned into the pGEM T-Easy vector, sequenced and compared to NCBIdatabase sequence, and finally cloned into the pet23b vector (EMDMillipore) in frame with the 6× His tag. This vector was thentransfected into Rosetta e.coli (EMD Millipore), grown and induced with0.4 mM IPTG (Invitrogen) to express Galectin-1. Galectin-1 was thenpurified as described in the pet vector handbook using the nickel Talon(Clontech) column and imidazole (Sigma-Aldrich) buffer for elution.Purified Galectin-1 was then dialyzed in PBS and used in variousstudies.

Tissue Culture. C2C12 myoblasts and myotubes were grown as previouslydescribed (Rooney PNAS 2009). α7βgal +/− myoblasts were originallyisolated and maintained as described (Rooney PNAS 2009). Briefly,myoblasts were grown and maintained in DMEM without phenol red (GIBCO,Grand Island, NY), 20% FBS (Atlanta Biologicals, Lawrenceville, Ga.),0.5% chick-embryo extract (CEE, Seralab, West Sussex, UK), 1%L-glutamine (GIBCO, Grand Island, NY) and 1% penicillin/streptomycin(PS) (GIBCO, Grand Island, NY). All myoblasts were maintained below 70%confluence until use in assay. Myoblasts were differentiated intomyotubes in DMEM without phenol red (GIBCO, Grand Island, NY), 1%horse-serum, and 1% Penicillin/Streptomycin (P/S)+L-Glutamine All cellswere maintained in TC incubators at 37° C. with 5% CO2. Myoblast α7Integrin drug enhancement assay. A total of 5000 α7βgal^(−/−)myoblastswere dispensed in 100 μL growth media using a 12-well multi-pippetteonto Nunc black sided TC coated 96-well plate. After 24 hours differentconcentrations of recombinant Galectin-1 were added to treatment wells,with identical amounts of PBS added as a negative control forcomparison. After incubating for 48 hours the media was aspirated, andcells were lysed with 50 μL of Mammalian Protein Extraction Reagent(MPER) per well followed by incubation at RT for 10 minutes.β-galactosidase (βgal) levels in each well were determined by adding 50μL of FDG solution (20% 0.1M Sodium phosphate buffer pH 7.0 (Sigma),0.2% 1 M mgC12 (Sigma), 0.2% 20 mM Fluorescein di-galactoside (FDG)(Marker Gene Technologies), and 79.6% dH2O) and incubating for 20minutes at RT in the dark. 100 μL/well stop solution (2× TE) was thenadded and plates were read for fluorescence on the Victor V(Perkin-Elmer) with an excitation filter at 485 nm, an emission filterat 535 nm, and a 0.1 s/well count time.

Galectin Treatment of C2C12 myoblasts. C2C12 myoblasts were treated withdifferent amounts of recombinant Galectin-1 for 48 hours, washed in PBS,and lysed in either 1xRIPA with protease inhibitor cocktail for westernblotting or in Trizol (Invitrogen) for quantitative real-time PCR.Intramuscular (IM) Tibialus anterior (TA) treatments. Between 20 ng and150 μg of Galectin-1 was delivered into the left mouse TA muscle by IMinjection with an equal volume of PBS delivered to the right. Mice werethen sacrificed 48 hours later and the TA muscles were removed for usein other studies.

Intraperitoneal (IP) Galectin-1 treatments: Treatment of mice wasstarted at 10 days of age either weekly or bi-weekly with 5 mg/kg or 20mg/kg of recombinant Galectin-1 and with corresponding volume of PBS ascontrols. All treatments were well tolerated with no observedside-effects and positive results on muscle and bone. Mouse gripstrength was determined using a tensometer as previously described(Rooney, PNAS 2009, which is hereby incorporated by reference in itsentirety).

Western Blotting. Protein concentrations of extracts from myoblast ormouse gastrocnemius tissue (protein extracted in RIPA + Prot Inhibs)were analyzed by BCA (Pierce) and then loaded at identicalconcentrations into SDS-PAGE gels and run under standard conditions.Proteins were then transferred to nitrocellulose and probed using therabbit polyclonal or mouse monoclonal antibodies against α7a Integrin,α7B Integrin, Galectin-1 (AbNova H00003956-D01P), β1D Integrin,α-dystroglycan, 3-dystroglycan H-242 (sc-28535), β-sarcoglycan H-98(sc-28279), γ-sarcoglycan Z-24 (sc-133984), δ-sarcoglycan H-55(sc-28281), ε-sarcoglycan H-67 (sc-28282, all sc antibodies are fromSantaCruz Biotechnology), and sarcospan.

Quantitative real-time PCR (qRTPCR). Total RNA from powdered mouse TAmuscle or myoblasts was isolated using Trizol (Invitrogen, Grand Island,N.Y.) followed by DNase treatment (Promega, Madison, Wis.), and cDNA wasmade with random hexamers (IDTDNA) and Superscript III (Invitrogen,Grand Island, N.Y.) using standard procedures. Quantitative real-timePCR was performed using Quanta Perfecta SYBR-Green with ROX Master Mixand was run and analyzed as previously described (Doe et al., J. CellScience: 124: 2287-2297, 2011). Primers against ITGA7, ITGB 1, andLGALS1 were described in Doe et al, (Id.).

Crysectioning and histology. 10μm sections of TA muscles from mice wereobtained using a Leica Cryostat. Hemotoxylin and eosin (H&E) stainingwas performed using standard procedures and images were taken using anOlympus Fluoview FV1000 Laser Confocal Microscope. Centrally locatednuclei counts and minimum Ferrets diameter measurements were performedusing standard procedures.

Mouse digital radiography. Digital radiography was performed on 5-weekand 10-week old mdx mice using a Sound-eklin tru/Digital radiographymachine. Femur, lower jaw, and tibia length measurements involved theuse of both Sound-eklin eSeries software and Image J and were simpleline/curve length measurements from the radiograph. Spinal curvature(kyphosis) was analyzed by drawing a line from the base of the spine atthe neck to the base of the spine at the beginning of the hip bone, Aperpendicular line was then drawn from the apex of the spinal curve andthe length of this line was used to measure kyphosis.

Statistical analysis. All statistical analysis was performed usingGraphPad Prism 5 software. Averaged data are reported as the mean ± thestandard error of the mean (s.e.m.). Comparison for two groups wasperformed using a Student's t-test and between multiple groups usingKruskal-Wallis one-way ANOVA on ranks for nonparametric data. P<0.05 wasconsidered statistically significant.

ii. Results

FIGS. 15A-15D demonstrate Galectin-1 treatment of myoblasts and myotubesleads to elevated levels of α7 and β1 Integrins at both the transcriptand protein levels. FIGS. 16A-16D show intermuscular (IM) injections ofmdx mouse tibialus anterior (TA) muscles with recombinant Galectin-1reduced muscle damage and the need for regeneration as determined by thehistological appearance of centrally located nuclei (CLN). Galectin-1treatment of mdx mice was also found to increase protein levels ofmembers of the sarcolemmal stabilizing dystroglycan complex (DGC) whichare normally lost in the absence of dytrophin (see FIGS. 17A-17J).Galectin-1 treatment of mdx mice increased transcript levels of membersof the α7β1 Integrin complex and LGALS1 (see FIGS. 18A-18C). FIGS.19A-19D are graphs illustrating galectin-1 treatment of mdx miceincreasd relative strength, decreases fatigue, and normalizes musclehistological fiber size. Galectin-1 treatment of mdx mice increasedmuscle strength which prevented kyphosis in 10-week old mice (FIG. 20).Further, Galectin-1 treatment of mdx mice increased bone growth duringdevelopment (see FIGS. 21A-21F). These studies clearly demonstrate thetherapeutic activities of Galectin-1 and in particular, the ability ofGalectin-1 to increase muscle strength, bone growth, decrease muscledamage, and decrease muscle fatigue.

In view of the many possible embodiments to which the principles of thedisclosed invention may be applied, it should be recognized that theillustrated embodiments are only preferred examples of the invention andshould not be taken as limiting the scope of the invention. Rather, thescope of the invention is defined by the following claims. We thereforeclaim as our invention all that comes within the scope and spirit ofthese claims.

1. (canceled)
 2. A method of increasing or maintaining muscle strength,muscle density, and/or bone density in a subject, comprisingadministering an effective amount of a Galectin-1 composition,Galectin-3 composition or a combination thereof to the subject in needthereof, thereby increasing or maintaining muscle strength, muscledensity, and/or bone density.
 3. A method of preventing, inhibitingand/or reducing muscle injury or loss and/or bone loss in a subject,comprising administering an effective amount of a Galectin-1composition, Galectin-3 composition or a combination thereof to thesubject in need thereof, thereby preventing, inhibiting, and/orreducing, muscle injury or loss and/or bone loss.
 4. The method of claim2, wherein the method includes administering an effective amount of aGalectin-1 composition.
 5. The method of claim 4, wherein the Galectin-1composition comprises Galectin-1 or fragments thereof and a carrier. 6.The method of claim 5, wherein the Galectin-1 composition isadministered by systemic administration.
 7. The method of claim 5,wherein the Galectin-1 composition is administered intramuscularly. 8.The method of claim 5, wherein the Galectin-1 composition isadministered intraperitoneally.
 9. The method of claim 5, wherein thesubject has or is at risk of acquiring a muscular dystrophy.
 10. Themethod of claim 9, wherein the subject has or is at risk of acquiringDuchenne muscular dystrophy.
 11. The method of claim 2, wherein themethod comprises administering an effective amount of a Galectin-1composition to a subject that has or is at risk of muscle/bone injury ormuscle/bone loss, such as an athlete or astronaut.
 12. The method ofclaim 2, wherein the method comprises administering an effective amountof a Galectin-1 composition to a subject at risk of acquiring orsuffering from kyphosis, muscular dystrophies, scoliosis, a broken bone,a fractured bone, muscle strain, muscle tear, tendon injury,osteoporosis, rheumatoid arthritis, lupus erythematosus, scoliosisand/or multiple sclerosis.
 13. The method of claim 2, wherein the methodcomprises administering an effective amount of a Galectin-1 compositionto a subject prior to and/or following a surgical procedure.
 14. Themethod of claim 2, wherein the method comprises administering aneffective amount of a Galectin-1 composition to a subject prior to,during and/or following pregnancy.
 15. The method of claim 2, whereinthe method comprises administering an effective amount of a Galectin-1composition to a short or long-term coma subject.
 16. The method ofclaim 3, wherein the method includes administering an effective amountof a Galectin-1 composition.
 17. The method of claim 16, wherein theGalectin-1 composition comprises Galectin-1 or fragments thereof and acarrier.
 18. The method of claim 17, wherein the Galectin-1 compositionis administered by systemic, intramuscular or intraperitonealadministration.
 19. The method of claim 16, wherein the subject has oris at risk of acquiring a muscular dystrophy.
 20. The method of claim19, wherein the subject has or is at risk of acquiring Duchenne musculardystrophy.